The purpose of this essay is threefold: to give an outline of the life and the various achievements of Theodor Escherich, to provide a background to his discovery of what he called Bacterium coli commune (now Escherichia coli), and to indicate the enormous impact of studies with this organism, long before it became the cornerstone of research in bacteriology and in molecular biology.

Citation: Agnes Ullmann. escherichia coli and the french school of molecular biology. doi:10.1128/ecosalplus.1.1.1

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André Lwoff, Jacques Monod, and François Jacob, the leaders of the French school of molecular biology, greatly contributed between 1937 and 1965 to its development and triumph. The main discovery of Lwoff was the elucidation of the mechanism of bacteriophage induction, the phenomenon of lysogeny, that led to the model of genetic regulation uncovered later by Jacob and Monod. Working on bacterial growth, Monod discovered in 1941 the phenomenon of diauxy and uncovered the nature of enzyme induction. By combining genetic and biochemical approaches, Monod brought to light the structure and functions of the Escherichia coli lactose system, comprising the genes necessary for lactose metabolism, i.e., β-galactosidase and lactose permease, a pump responsible for accumulation of galactosides into the cells. An additional genetic factor (the i gene) determines the inducibility and constitutivity of enzyme synthesis. Around the same time, François Jacob and Elie Wollman dissected the main events of bacterial conjugation that enabled them to construct a map of the E. coli chromosome and to demonstrate its circularity. The genetic analysis of the lactose system led Monod and Jacob to elucidate the mechanism of the regulation of gene expression and to propose the operon model: a unit of coordinate transcription. One of the new concepts that emerged from the operon model was messenger RNA. In 1963, Monod developed one of the most elegant concepts of molecular biology, the theory of allostery. In 1965, Lwoff, Monod and Jacob were awarded the Nobel Prize in Physiology or Medicine.

The creation of the "Phage group" by M. Delbrück, S. E. Luria, and A. D. Hershey in 1940 at Cold Spring Harbor played a crucial role in the development of molecular biology. In the 1940s, working with Escherichia coli and its viruses, Luria and Delbrück discovered the spontaneous nature of bacterial mutations and Hershey described recombination in bacteriophages and demonstrated with M. Chase that the genetic material that infects bacteria is DNA. At the same time, S. Benzer defined the structure of a functional genetic unit and J. Lederberg and E. Tatum discovered sexual recombination between bacteria. Some years later, Lederberg's group discovered extrachromosomal particles, the plasmids, and a novel way of genetic transfer through bacteriophages, called transduction. In 1949, at the Pasteur Institute in Paris, A. Lwoff uncovered the mechanism of lysogeny. Shortly afterwards, F. Jacob and E. Wollman unraveled the mechanism of the sexual process in E. coli and established the circularity of the bacterial chromosome. In the 1960s, J. Monod and F. Jacob, by genetic analysis of the E. coli lactose system, proposed the operon model for gene regulation and introduced the concept of messenger RNA. The elucidation of the double helix structure of DNA in 1953 by F. Crick and J. Watson had major consequences: the establishment of the copying mechanism (Meselson and Stahl), the discovery of the nature of the genetic code (S. Brenner) leading to its deciphering. E. coli and its phages were instrumental in the development of recombinant DNA technology based on the discovery of the restriction-modification system by W. Arber.

The Golden Age of Phage Research, where phage was the favored material for attacking many basic questions in molecular biology, lasted from about 1940 to 1970. The era was initiated by Ellis and Delbrück, whose analysis defined the relevant parameters to measure in studying phage growth, and depended on the fact that the contents of a plaque can comprise descendants of a single infecting particle. It ended around 1970 because definitive methods had then become available for answering the same questions in other systems. Some of the accomplishments of phage research were the demonstration by Hershey and Chase that the genetic material of phage T2 is largely composed of DNA, the construction of linkage maps of T2 and T4 by Hershey and Rotman and their extension to very short molecular distances by Benzer, and the isolation of conditionally lethal mutants in T4 by Epstein et al. and in λ by Campbell. The dissection of the phage life cycle into causal chains was explored by Edgar and Wood for T4 assembly and later in the regulation of lysogeny by Kaiser, extended to the molecular level by Ptashne and others. Restriction/modification was discovered in λ by Bertani and Weigle, and the biochemical mechanism was elucidated by Arber and by Smith.

In 1995, an editorial in Science (267:1575) commented that predictions made some 25 years previously regarding “Biology and the Future of Man” were largely fulfilled but that “the most revolutionary and unexpected findings were not predicted.” We would be glad to do as well! As we stated at the beginning, our work as editors of the Escherichia coli and Salmonella book did not endow us with special powers of prophecy but it does permit us to express our excitement for the future. In our opinion, E. coli and S. enterica will continue to play a central role in biological research. This is not because they are intrinsically more interesting than any other bacteria, as we believe that all bacteria are equally interesting. However, knowledge builds on knowledge, and it is here that these two species continue to have a large edge not only over other microorganisms but also, for some time to come, over all other forms of life. It is interesting in this connection that biotechnology, having made detours through other microorganisms, always seems to return to E. coli.

The history of Shigella, the causative agent of bacillary dysentery, is a long and fascinating one. This brief historical account starts with descriptions of the disease and its impact on human health from ancient time to the present. Our story of the bacterium starts just before the identification of the dysentery bacillus by Kiyoshi Shiga in 1898 and follows the scientific discoveries and principal scientists who contributed to the elucidation of Shigella pathogenesis in the first 100 years. Over the past century, Shigella has proved to be an outstanding model of an invasive bacterial pathogen and has served as a paradigm for the study of other bacterial pathogens. In addition to invasion of epithelial cells, some of those shared virulence traits include toxin production, multiple-antibiotic resistance, virulence genes encoded on plasmids and bacteriophages, global regulation of virulence genes, pathogenicity islands, intracellular motility, remodeling of host cytoskeleton, inflammation/polymorphonuclear leukocyte signaling, apoptosis induction/inhibition, and “black holes” and antivirulence genes. While there is still much to learn from studying Shigella pathogenesis, what we have learned so far has also contributed greatly to our broader understanding of bacterial pathogenesis.

Proteinaceous, nonflagellar surface appendages constitute a variety of structures, including those known variably as fimbriae or pili. Constructed by distinct assembly pathways resulting in diverse morphologies, fimbriae have been described to mediate functions including adhesion, motility, and DNA transfer. As these structures can represent major diversifying elements among Escherichia and Salmonella isolates, multiple fimbrial classification schemes have been proposed and a number of mechanistic insights into fimbrial assembly and function have been made. Herein we describe the classifications and biochemistry of fimbriae assembled by the chaperone/usher, curli, and type IV pathways.

Fimbria-mediated interaction with the host elicits both innate and adaptive immune responses, and thus their expression may not always be beneficial in vivo. Furthermore, the metabolic drain of producing fimbriae is significant. It is not surprising, therefore, to find that fimbrial production in Escherichia coli and Salmonella enterica is under extensive environmental regulation. In many instances, fimbrial expression is regulated by phase variation, in which individual cells are capable of switching between fimbriate and afimbriate states to produce a mixed population. Mechanisms of phase variation vary considerably between different fimbriae and involve both genetic and epigenetic processes. Notwithstanding this, fimbrial expression is also sometimes controlled at the posttranscriptional level. In this chapter, we review key features of the regulation of fimbrial gene expression in E. coli and Salmonella. The occurrence and distribution of fimbrial operons vary significantly among E. coli pathovars and even among the many Salmonella serovars. Therefore, general principles are presented on the basis of detailed discussion of paradigms that have been extensively studied, including Pap, type 1 fimbriae, and curli. The roles of operon specific regulators like FimB or CsgD and of global regulatory proteins like Lrp, CpxR, and the histone-like proteins H-NS and IHF are reviewed as are the roles of sRNAs and of signalling nucleotide cyclic-di-GMP. Individual examples are discussed in detail to illustrate how the regulatory factors cooperate to allow tight control of expression of single operons. Molecular networks that allow coordinated expression between multiple fimbrial operons and with flagella in a single isolate are also presented. This chapter illustrates how adhesin expression is controlled, and the model systems also illustrate general regulatory principles germane to our overall understanding of bacterial gene regulation.

The capsule is a cell surface structure composed of long-chain polysaccharides that envelops many isolates of Escherichia coli. It protects the cell against host defenses or physical environmental stresses, such as desiccation. The component capsular polysaccharides (CPSs) are major surface antigens in E. coli. They are named K antigens (after the German word Kapsel). Due to variations in CPS structures, more than 80 serologically unique K antigens exist in E. coli. Despite the hypervariability in CPS structures, only two capsule-assembly strategies exist in E. coli. These have led to the assignment of group 1 and group 2 capsules, and many of the key elements of the corresponding assembly pathways have been resolved. Structural features, as well as genetic and regulatory variations, give rise to additional groups 3 and 4. These employ the same biosynthesis processes described in groups 2 and 1, respectively. Each isolate possesses a distinctive set of cytosolic and inner-membrane enzymes, which generate a precise CPS structure, defining a given K serotype. Once synthesized, a multiprotein complex is needed to translocate the nascent CPS across the Gram-negative cell envelope to the outer surface of the outer membrane, where the capsule structure is assembled. While the translocation machineries for group 1 and group 2 CPSs are fundamentally different from one another, they possess no specificity for a given CPS structure. Each is conserved in all isolates producing capsules belonging to a particular group.

Outer membrane vesicles (blebs) are produced by Escherichia coli, Salmonella, and all other gram-negative bacteria both in vitro and in vivo. Most of the research in the field has focused on the properties of vesicles derived from pathogenic bacteria and their interactions with eukaryotic cells. These data indicate that vesicles are able to contribute to pathogenesis. Thus, it appears that pathogenic gram-negative bacteria have co-opted vesicles for the dissemination of virulence determinants. However, the role of vesicle production by nonpathogenic bacteria is less obvious. This section reviews the data demonstrating the mechanistic and physiological basis of outer membrane vesicle production by bacteria. Vesiculation can be seen as a mechanism for cells to react to conditions in the surrounding environment by carrying away unnecessary components and allowing rapid modification of the outer membrane composition. In addition, vesicles can transmit biological activities distant from the originating cell. Vesicles could act to bind and deplete host immune factors at the site of infection that would otherwise attack the bacteria. Vesicles in the area surrounding the cell may also provide the cell protection inside a human or animal host. The concept of vesicles as virulence factors has received considerable attention, and they are likely to play a significant role in the pathogenesis of gram-negative bacteria. By analysis of their composition, mechanism of formation, regulation, and physiological function, progress is being made in understanding the ubiquitous nature of outer membrane vesicles produced by gram-negative bacteria.

This review provides a brief review of the current understanding of the structure-function relationship of the Escherichia coli nucleoid developed after the overview by Pettijohn focusing on the physical properties of nucleoids. Isolation of nucleoids requires suppression of DNA expansion by various procedures. The ability to control the expansion of nucleoids in vitro has led to purification of nucleoids for chemical and physical analyses and for high-resolution imaging. Isolated E. coli genomes display a number of individually intertwined supercoiled loops emanating from a central core. Metabolic processes of the DNA double helix lead to three types of topological constraints that all cells must resolve to survive: linking number, catenates, and knots. The major species of nucleoid core protein share functional properties with eukaryotic histones forming chromatin; even the structures are different from histones. Eukaryotic histones play dynamic roles in the remodeling of eukaryotic chromatin, thereby controlling the access of RNA polymerase and transcription factors to promoters. The E. coli genome is tightly packed into the nucleoid, but, at each cell division, the genome must be faithfully replicated, divided, and segregated. Nucleoid activities such as transcription, replication, recombination, and repair are all affected by the structural properties and the special conformations of nucleoid. While it is apparent that much has been learned about the nucleoid, it is also evident that the fundamental interactions organizing the structure of DNA in the nucleoid still need to be clearly defined.

Citation: Hans Bremer, Patrick P. Dennis. Modulation of chemical composition and other parameters of the cell at different exponential growth rates. doi:10.1128/ecosal.5.2.3

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This review begins by briefly presenting the history of research on the chemical composition and other parameters of cells of E. coli and S. enterica at different exponential growth rates. Studies have allowed us to determine the in vivo strength of promoters and have allowed us to distinguish between factor-dependent transcriptional control of the promoter and changes in promoter activity due to changes in the concentration of free functional RNA polymerase associated with different growth conditions. The total, or bulk, amounts of RNA and protein are linked to the growth rate, because most bacterial RNA is ribosomal RNA (rRNA). Since ribosomes are required for protein synthesis, their number and their rate of function determine the rate of protein synthesis and cytoplasmic mass accumulation. Many mRNAs made in the presence of amino acids have strong ribosome binding sites whose presence reduces the expression of all other active genes. This implies that there can be profound differences in the spectrum of gene activities in cultures grown in different media that produce the same growth rate. Five classes of growth-related parameters that are generally useful in describing or establishing the macromolecular composition of bacterial cultures are described in detail in this review. A number of equations have been reported that describe the macromolecular composition of an average cell in an exponential culture as a function of the culture doubling time and five additional parameters: the C- and D-periods, protein per origin (PO), ribosome activity, and peptide chain elongation rate.

Domain 3

Classically, metabolism was investigated by studying molecular characteristics of enzymes and their regulators in isolation. This reductionistic approach successfully established mechanistic relationships with the immediate interacting neighbors and allowed reconstruction of network structures. Severely underdeveloped was the ability to make precise predictions about the integrated operation of pathways and networks that emerged from the typically nonlinear and complex interactions of proteins and metabolites. The burden of metabolic engineering is a consequence of this fact—one cannot yet predict with any certainty precisely what needs to be engineered to produce more complex phenotypes. What was and still is missing are concepts, methods, and algorithms to integrate data and information into a quantitatively coherent whole, as well as theoretical concepts to reliably predict the consequence of environmental stimuli or genetic interventions. This introduction and perspective to Domain 3, Metabolism and Metabolic Fluxes, starts with a brief overview of the panoply of global measurement technologies that herald the dawning of systems biology and whose impact on metabolic research is apparent throughout the Domain 3. In the middle section, applications to Escherichia coli are used to illustrate general concepts and successes of computational methods that approach metabolism as a network of interacting elements, and thus have potential to fill the gap in quantitative data and information integration. The final section highlights prospective focus areas for future metabolic research, including functional genomics, eludication of evolutionary principles, and the integration of metabolism with regulatory networks.

Escherichia coli contains a versatile respiratory chain that oxidizes 10 different electron donor substrates and transfers the electrons to terminal reductases or oxidases for the reduction of six different electron acceptors. Salmonella is able to use two more electron acceptors. The variation is further increased by the presence of isoenzymes for some substrates. A large number of respiratory pathways can be established by combining different electron donors and acceptors. The respiratory dehydrogenases use quinones as the electron acceptors that are oxidized by the terminal reductase and oxidases. The enzymes vary largely with respect to their composition, architecture, membrane topology, and the mode of energy conservation. Most of the energy-conserving dehydrogenases (FdnGHI, HyaABC, HybCOAB, and others) and the terminal reductases (CydAB, NarGHI, and others) form a proton potential (Δp) by a redox-loop mechanism. Two enzymes (NuoA-N and CyoABCD) couple the redox energy to proton translocation by proton pumping. A large number of dehydrogenases and terminal reductases do not conserve the redox energy in a proton potential. For most of the respiratory enzymes, the mechanism of proton potential generation is known or can be predicted. The H+/2e− ratios for most respiratory chains are in the range from 2 to 6 H+/2e−. The energetics of the individual redox reactions and the respiratory chains is described and related to the H+/2e− ratios.

The F1F0-ATP synthase (EC 3.6.1.34) is a remarkable enzyme that functions as a rotary motor. It is found in the inner membranes of Escherichia coli and is responsible for the synthesis of ATP in response to an electrochemical proton gradient. Under some conditions, the enzyme functions reversibly and uses the energy of ATP hydrolysis to generate the gradient. The ATP synthase is composed of eight different polypeptide subunits in a stoichiometry of α3β3γδεab2c10. Traditionally they were divided into two physically separable units: an F1 that catalyzes ATP hydrolysis (α3β3γδε) and a membrane-bound F0 sector that transports protons (ab2c10). In terms of rotary function, the subunits can be divided into rotor subunits (γεc10) and stator subunits (α3β3δab2). The stator subunits include six nucleotide binding sites, three catalytic and three noncatalytic, formed primarily by the β and α subunits, respectively. The stator also includes a peripheral stalk composed of δ and b subunits, and part of the proton channel in subunit a. Among the rotor subunits, the c subunits form a ring in the membrane, and interact with subunit a to form the proton channel. Subunits γ and ε bind to the c-ring subunits, and also communicate with the catalytic sites through interactions with α and β subunits. The eight subunits are expressed from a single operon, and posttranscriptional processing and translational regulation ensure that the polypeptides are made at the proper stoichiometry. Recent studies, including those of other species, have elucidated many structural and rotary properties of this enzyme.

The number of NADH dehydrogenases and their role in energy transduction in Escherchia coli have been under debate for a long time. Now it is evident that E. coli possesses two respiratory NADH dehydrogenases, or NADH:ubiquinone oxidoreductases, that have traditionally been called NDH-I and NDH-II. This review describes the properties of these two NADH dehydrogenases, focusing on the mechanism of the energy converting NADH dehydrogenase as derived from the high resolution structure of the soluble part of the enzyme. In E. coli, complex I operates in aerobic and anaerobic respiration, while NDH-II is repressed under anaerobic growth conditions. The insufficient recycling of NADH most likely resulted in excess NADH inhibiting tricarboxylic acid cycle enzymes and the glyoxylate shunt. Salmonella enterica serovar Typhimurium complex I mutants are unable to activate ATP-dependent proteolysis under starvation conditions. NDH-II is a single subunit enzyme with a molecular mass of 47 kDa facing the cytosol. Despite the absence of any predicted transmembrane segment it has to be purified in the presence of detergents, and the activity of the preparation is stimulated by an addition of lipids.

Nitrate reduction to ammonia via nitrite occurs widely as an anabolic process through which bacteria, archaea, and plants can assimilate nitrate into cellular biomass. Escherichia coli and related enteric bacteria can couple the eight-electron reduction of nitrate to ammonium to growth by coupling the nitrate and nitrite reductases involved to energy-conserving respiratory electron transport systems. In global terms, the respiratory reduction of nitrate to ammonium dominates nitrate and nitrite reduction in many electron-rich environments such as anoxic marine sediments and sulfide-rich thermal vents, the human gastrointestinal tract, and the bodies of warm-blooded animals. This review reviews the regulation and enzymology of this process in E. coli and, where relevant detail is available, also in Salmonella and draws comparisons with and implications for the process in other bacteria where it is pertinent to do so. Fatty acids may be present in high levels in many of the natural environments of E. coli and Salmonella in which oxygen is limited but nitrate is available to support respiration. In E. coli, nitrate reduction in the periplasm involves the products of two seven-gene operons, napFDAGHBC, encoding the periplasmic nitrate reductase, and nrfABCDEFG, encoding the periplasmic nitrite reductase. No bacterium has yet been shown to couple a periplasmic nitrate reductase solely to the cytoplasmic nitrite reductase NirB. The cytoplasmic pathway for nitrate reduction to ammonia is restricted almost exclusively to a few groups of facultative anaerobic bacteria that encounter high concentrations of environmental nitrate.

Succinate and fumarate are four-carbon dicarboxylates that differ in the identity of their central bond (single or double). The oxidoreduction of these small molecules plays a central role in both aerobic and anaerobic respiration. During aerobic respiration, succinate is oxidized, donating two reducing equivalents, while in anaerobic respiration, fumarate is reduced, accepting two reducing equivalents. Two related integral membrane Complex II superfamily members catalyze these reactions, succinate:ubiquinone oxidoreductase (SQR) and fumarate:menaquinol oxidoreductase (QFR). The structure, function, and regulation of these integral-membrane enzymes are summarized here. The overall architecture of these Complex II enzymes has been found to consist of four subunits: two integral membrane subunits, and a soluble domain consisting of an iron-sulfur protein subunit, and a flavoprotein subunit. This architecture provides a scaffold that houses one active site in the membrane and another in the soluble milieu, making a linear electron transfer chain that facilities shuttling of reducing equivalents between the two active sites. A combination of kinetic measurements, mutagenesis, electron paramagnetic resonance spectroscopy, UV/Vis spectroscopy, and x-ray crystallography have suggested mechanisms for succinate:fumarate interconversion, electron transfer, and quinone:quinol interconversion. Of particular interest are the structural details that control directionality and make SQR and QFR primed for preferential catalysis each in different favored directions.

Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O2 is served by two major oxidoreductases (oxidases), cytochrome bo3 encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O2 as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo3 and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo3 and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C40). It has been proposed that the bo3 oxidase can have two ubiquinone-binding sites with different affinities.

“What’s new” in the revised article: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E. coli, although its spectral properties closely resemble those of cydABX-encoded cytochrome bd.

Escherichia coli is a versatile facultative anaerobe that can respire on a number of terminal electron acceptors, including oxygen, fumarate, nitrate, and S- and N-oxides. Anaerobic respiration using S- and N-oxides is accomplished by enzymatic reduction of these substrates by dimethyl sulfoxide reductase (DmsABC) and trimethylamine N-oxide reductase (TorCA). Both DmsABC and TorCA are membrane-associated redox enzymes that couple the oxidation of menaquinol to the reduction of S- and N-oxides in the periplasm. DmsABC is membrane bound and is composed of a membrane-extrinsic dimer with a 90.4-kDa catalytic subunit (DmsA) and a 23.1-kDa electron transfer subunit (DmsB). These subunits face the periplasm and are held to the membrane by a 30.8-kDa membrane anchor subunit (DmsC). The enzyme provides the scaffold for an electron transfer relay composed of a quinol binding site, five [4Fe-4S] clusters, and a molybdo-bis(molybdopterin guanine dinucleotide) (present nomenclature: Mo-bis-pyranopterin) (Mo-bisMGD) cofactor. TorCA is composed of a soluble periplasmic subunit (TorA, 92.5 kDa) containing a Mo-bis-MGD. TorA is coupled to the quinone pool via a pentaheme c subunit (TorC, 40.4 kDa) in the membrane. Both DmsABC and TorCA require system-specific chaperones (DmsD or TorD) for assembly, cofactor insertion, and/or targeting to the Tat translocon. In this chapter, we discuss the complex regulation of the dmsABC and torCAD operons, the poorly understood paralogues, and what is known about the assembly and translocation to the periplasmic space by the Tat translocon.

Two membranes enclose Gram-negative bacteria-an inner membrane consisting of phospholipid and an outer membrane having an asymmetric structure in which the inner leaflet contains phospholipid and the outer leaflet consists primarily of lipopolysaccharide. The impermeable nature of the outer membrane imposes a need for numerous outer membrane pores and transporters to ferry substances in and out of the cell. These outer membrane proteins have structures distinct from their inner membrane counterparts and most often function without any discernable energy source. In this chapter, we review the structures and functions of four classes of outer membrane protein: general and specific porins, specific transporters, TonB-dependent transporters, and export channels. While not an exhaustive list, these classes exemplify small-molecule transport across the outer membrane and illustrate the diversity of structures and functions found in Gram-negative bacteria.

Bacterial ion channels were known, but only in special cases, such as outer membrane porins in Escherichia coli and bacterial toxins that form pores in their target (bacterial or mammalian) membranes. The exhaustive coverage provided by a decade of bacterial genome sequencing has revealed that ion channels are actually widespread in bacteria, with homologs of a broad range of mammalian channel proteins coded throughout the bacterial and archaeal kingdoms. This review discusses four groups of bacterial channels: porins, mechano-sensitive (MS) channels, channel-forming toxins, and bacterial homologs of mammalian channels. The outer membrane (OM) of gram-negative bacteria blocks access of essential nutrients; to survive, the cell needs to provide a mechanism for nutrients to penetrate the OM. Porin channels provide this access by forming large, nonspecific aqueous pores in the OM that allow ions and vital nutrients to cross it and enter the periplasm. MS channels act as emergency release valves, allowing solutes to rapidly exit the cytoplasm and to dissipate the large osmotic disparity between the internal and external environments. MS channels are remarkable in that they do this by responding to forces exerted by the membrane itself. Some bacteria produce toxic proteins that form pores in trans, attacking and killing other organisms by virtue of their pore formation. The review focuses on those bacterial toxins that kill other bacteria, specifically the class of proteins called colicins. Colicins reveal the dangers of channel formation in the plasma membrane, since they kill their targets with exactly that approach.

This review reviews the ammonium/methylammonium transport (Amt) proteins of Escherichia coli and Salmonella enterica serovar Typhimurium. The Amt proteins and their homologs, the methylammonium/ammonium permease proteins of Saccharomyces cerevisiae, constitute a distinct class of membrane-associated ammonia transporters. Members of the Amt family are found in archaea, bacteria, fungi, plants, and invertebrate animals. In E. coli and serovar Typhimurium, the Amt proteins are essential to maintain maximal growth at low concentrations of ammonia, the preferred nitrogen source. Soupene and coworkers showed that a mutant of E. coli with only the low-affinity glutamate dehydrogenase pathway for assimilation of ammonia, which therefore grows slowly at low ammonia concentrations, is not relieved of its growth defect by overexpression of AmtB. A recent study on an Amt protein from tomato concluded that it was a specific transporter for NH4+. A trimeric stoichiometry for AmtB is supported by the observation of a direct interaction between AmtB and the trimeric signal-transduction protein GlnK. In E. coli, GlnK has been observed to associate with the membrane in an AmtB-dependent fashion. Both GlnK and GlnB are sensors of nitrogen status. Their interaction with AmtB suggests a role for AmtB in nitrogen regulation. In summary, AmtB is a membrane-associated ammonia transporter that is important for growth at external concentrations of the uncharged species (NH3) below about 50 nM. The preponderance of evidence suggests that AmtB specifically transports the charged species (NH4+) and that this transport is passive and, hence, bidirectional.

Maltose and maltodextrins are actively transported across the cytoplasmic membrane of Escherichia coli and Salmonella by a periplasmic binding protein (BP)- dependent transport system. Since 1996, there have been many advances in the understanding of the structure and mechanism of the maltose transporter, in the assembly of the membrane-associated transporter complex, and in the mechanism of regulation of transport both at the DNA and the protein level. The transporter has been studied in detergent and reconstituted in liposome vesicles, and while many features, including the ability of maltose-binding protein (MBP) to stimulate ATPase activity, are retained in detergent, it has been noted that the basal ATPase activity of the transporter is elevated in detergent compared with liposomes. This review focuses on these recent developments, which have culminated in a high resolution structure of MBP in a complex with the MalFGK2 transporter. While this review focuses on the maltose system, complementary work has been carried out on many different ATP binding cassette (ABC) transporters, all of which has contributed in important ways to the understanding of the maltose transport system. The regulation of the maltose transport system, at the DNA level, is implemented by the synergistic action of MalT and cAMP/CAP complex and, at the protein level, by interactions of MalK with unphosphorylated EIIAglc, a signal-transducing component of the phosphoenolpyruvate-glucose phosphotransferase system.

An interesting model for studying environmental influences shaping microbial evolution is provided by a multitude of copper resistance and copper homeostasis determinants in enteric bacteria. This review describes these determinants and tries to relate their presence to the habitat of the respective organism, as a current hypothesis predicts that the environment should determine an organism’s genetic makeup. In Escherichia coli there are four regulons that are induced in the presence of copper. Two, the CueR and the CusR regulons, are described in detail. A central component regulating intracellular copper levels, present in all free-living enteric bacteria whose genomes have so far been sequenced, is a Cu(I)translocating P-type ATPase. The P-type ATPase superfamily is a ubiquitous group of proteins involved in the transport of charged substrates across biological membranes. Whereas some components involved in copper homeostasis can be found in both anaerobes and aerobes, multi-copper oxidases (MCOs) implicated in copper tolerance in E. coli, such as CueO and the plasmid-based PcoA, can be found only in aerobic organisms. Several features indicate that CueO, PcoA, and other related MCOs are specifically adapted to combat copper-mediated oxidative damage. In addition to these well-characterized resistance operons, there are numerous other genes that appear to be involved in copper binding and trafficking that have not been studied in great detail. SilE and its homologue PcoE, for example, are thought to effect the periplasmic binding and sequestration of silver and copper, respectively.

This review reviews the properties and regulation of the Salmonella enterica serovar Typhimurium and Escherichia coli transporters that mediate Mg2+ influx: CorA and the Mgt P-type ATPases. In addition, potential Mg2+ regulation of transcription and translation, largely via the PhoPQ two component system, is discussed. CorA proteins are a unique class of transporters and are widespread in the Bacteria and Archaea, with rather distant but functional homologs in eukaryotes. The Mgt transporters are highly homologous to other P-type ATPases but are more closely related to the eukaryotic H+ and Ca2+ ATPases than to most prokaryotic ATPases. Hundreds of homologs of CorA are currently known from genomic sequencing. In contrast, only when extracellular and possibly intracellular Mg2+ levels fall significantly is the expression of mgtA and mgtB induced. Topology studies using blaM and lacZ fusions initially indicated that the Salmonella serovar Typhimurium CorA contained three transmembrane (TM) segments; however, subsequent data obtained using a variety of approaches showed that the CorA superfamily of proteins have only two TMs at the extreme C terminus. PhoP-PhoQ is a two-component system consisting of PhoQ, the sensor/receptor histidine kinase, and PhoP, the response regulator/transcriptional activator. The expression of both mgtA and mgtCB in either E. coli or Salmonella serovar Typhimurium is markedly induced in a PhoPQ-dependent manner by low concentrations of Mg2+ in the medium. phoP and phoQ form an operon with two promoters in both E. coli and Salmonella serovar Typhimurium.

This chapter focuses on transition metals. All transition metal cations are toxic—those that are essential for Escherichia coli and belong to the first transition period of the periodic system of the element and also the "toxic-only" metals with higher atomic numbers. Common themes are visible in the metabolism of these ions. First, there is transport. High-rate but low-affinity uptake systems provide a variety of cations and anions to the cells. Control of the respective systems seems to be mainly through regulation of transport activity (flux control), with control of gene expression playing only a minor role. If these systems do not provide sufficient amounts of a needed ion to the cell, genes for ATP-hydrolyzing high-affinity but low-rate uptake systems are induced, e.g., ABC transport systems or P-type ATPases. On the other hand, if the amount of an ion is in surplus, genes for efflux systems are induced. By combining different kinds of uptake and efflux systems with regulation at the levels of gene expression and transport activity, the concentration of a single ion in the cytoplasm and the composition of the cellular ion "bouquet" can be rapidly adjusted and carefully controlled. The toxicity threshold of an ion is defined by its ability to produce radicals (copper, iron, chromate), to bind to sulfide and thiol groups (copper, zinc, all cations of the second and third transition period), or to interfere with the metabolism of other ions. Iron poses an exceptional metabolic problem due its metabolic importance and the low solubility of Fe(III) compounds, combined with the ability to cause dangerous Fenton reactions. This dilemma for the cells led to the evolution of sophisticated multi-channel iron uptake and storage pathways to prevent the occurrence of unbound iron in the cytoplasm. Toxic metals like Cd2+ bind to thiols and sulfide, preventing assembly of iron complexes and releasing the metal from iron-sulfur clusters. In the unique case of mercury, the cation can be reduced to the volatile metallic form. Interference of nickel and cobalt with iron is prevented by the low abundance of these metals in the cytoplasm and their sequestration by metal chaperones, in the case of nickel, or by B12 and its derivatives, in the case of cobalt. The most dangerous metal, copper, catalyzes Fenton-like reactions, binds to thiol groups, and interferes with iron metabolism. E. coli solves this problem probably by preventing copper uptake, combined with rapid efflux if the metal happens to enter the cytoplasm.

Escherichia coli and Salmonella enterica serovar Typhimurium exhibit a remarkable versatility in the usage of different sugars as the sole source of carbon and energy, reflecting their ability to make use of the digested meals of mammalia and of the ample offerings in the wild. Degradation of sugars starts with their energy-dependent uptake through the cytoplasmic membrane and is carried on further by specific enzymes in the cytoplasm, destined finally for degradation in central metabolic pathways. As variant as the different sugars are, the biochemical strategies to act on them are few. They include phosphorylation, keto-enol isomerization, oxido/reductions, and aldol cleavage. The catabolic repertoire for using carbohydrate sources is largely the same in E. coli and in serovar Typhimurium. Nonetheless, significant differences are found, even among the strains and substrains of each species. We have grouped the sugars to be discussed according to their first step in metabolism, which is their active transport, and follow their path to glycolysis, catalyzed by the sugar-specific enzymes. We will first discuss the phosphotransferase system (PTS) sugars, then the sugars transported by ATP-binding cassette (ABC) transporters, followed by those that are taken up via proton motive force (PMF)-dependent transporters. We have focused on the catabolism and pathway regulation of hexose and pentose monosaccharides as well as the corresponding sugar alcohols but have also included disaccharides and simple glycosides while excluding polysaccharide catabolism, except for maltodextrins.

Following elucidation of the regulation of the lactose operon in Escherichia coli, studies on the metabolism of many sugars were initiated in the early 1960s. The catabolic pathways of D-gluconate and of the two hexuronates, D-glucuronate and D-galacturonate, were investigated. The post genomic era has renewed interest in the study of these sugar acids and allowed the complete characterization of the D-gluconate pathway and the discovery of the catabolic pathways for L-idonate, D-glucarate, galactarate, and ketogluconates. Among the various sugar acids that are utilized as sole carbon and energy sources to support growth of E. coli, galacturonate, glucuronate, and gluconate were shown to play an important role in the colonization of the mammalian large intestine. In the case of sugar acid degradation, the regulators often mediate negative control and are inactivated by interaction with a specific inducer, which is either the substrate or an intermediate of the catabolism. These regulators coordinate the synthesis of all the proteins involved in the same pathway and, in some cases, exert crosspathway control between related catabolic pathways. This is particularly well illustrated in the case of hexuronide and hexuronate catabolism. The structural genes encoding the different steps of hexuronate catabolism were identified by analysis of numerous mutants affected for growth with galacturonate or glucuronate. E. coli is able to use the diacid sugars D-glucarate and galactarate (an achiral compound) as sole carbon source for growth. Pyruvate and 2-phosphoglycerate are the final products of the D-glucarate/galactarate catabolism.

The metabolic connection between glycerol and methylglyoxal (MG) is principally that DHAP, which is an intermediate in the aerobic breakdown of glycerol, is also the major precursor of MG, being the substrate for methylglyoxal synthase (MGS). The synthesis of MG is a consequence of unbalanced metabolism related either to a limitation for phosphate or to excessive carbon flux through the pathways that have the capacity to generate significant pools of DHAP. Cells producing MG produce a poison as an intermediate strategy for survival of metabolic imbalance. Indeed the panoply of metabolic regulation in this sector of catabolism can be seen as a strategy to avoid death by self-poisoning. Glycerol entry into Escherichia coli and Salmonella enterica serovar Typhimurium is facilitated by the aquaglyceroporin, GlpF. A homologous protein in serovar Typhimurium, PduF, facilitates the entry of 1,2-propanediol (Ppd) and is part of the Ppd metabolic pathway. MGS catalyzes the elimination of phosphate from DHAP, forming an enzyme-bound enediol(ate) intermediate that is released from the enzyme, followed by release of inorganic phosphate. The enzyme is highly specific for DHAP. Multiple MG detoxification pathways are found in both E. coli and serovar Typhimurium, but the dominant pathway is the GSH-dependent glyoxalase III system. The KefB and KefC systems have evolved to provide protection during detoxification of electrophiles. KefB and KefC are GSH-gated K+ efflux systems that are activated by the formation and binding of glutathione adducts that are generated during detoxification.

This review concerns the uptake and degradation of those molecules that are wholly or largely converted to acetyl-coenzyme A (CoA) in the first stage of metabolism in Escherichia coli and Salmonella enterica. These include acetate, acetoacetate, butyrate and longer fatty acids in wild type cells plus ethanol and some longer alcohols in certain mutant strains. Entering metabolism as acetyl-CoA has two important general consequences. First, generation of energy from acetyl-CoA requires operation of both the citric acid cycle and the respiratory chain to oxidize the NADH produced. Hence, acetyl-CoA serves as an energy source only during aerobic growth or during anaerobic respiration with such alternative electron acceptors as nitrate or trimethylamine oxide. In the absence of a suitable oxidant, acetyl-CoA is converted to a mixture of acetic acid and ethanol by the pathways of anaerobic fermentation. Catabolism of acetyl-CoA via the citric acid cycle releases both carbon atoms of the acetyl moiety as carbon dioxide and growth on these substrates as sole carbon source therefore requires the operation of the glyoxylate bypass to generate cell material. The pair of related two-carbon compounds, glycolate and glyoxylate are also discussed. However, despite having two carbons, these are metabolized via malate and glycerate, not via acetyl-CoA. In addition, mutants of E. coli capable of growth on ethylene glycol metabolize it via the glycolate pathway, rather than via acetyl- CoA. Propionate metabolism is also discussed because in many respects its pathway is analogous to that of acetate. The transcriptional regulation of these pathways is discussed in detail.

C4-dicarboxylates and the C4-dicarboxylic amino acid l-aspartate support aerobic and anaerobic growth of Escherichia coli and related bacteria. In aerobic growth, succinate, fumarate, D- and L-malate, L-aspartate, and L-tartrate are metabolized by the citric acid cycle and associated reactions. Because of the interruption of the citric acid cycle under anaerobic conditions, anaerobic metabolism of C4-dicarboxylates depends on fumarate reduction to succinate (fumarate respiration). In some related bacteria (e.g., Klebsiella), utilization of C4-dicarboxylates, such as tartrate, is independent of fumarate respiration and uses a Na+-dependent membrane-bound oxaloacetate decarboxylase. Uptake of the C4-dicarboxylates into the bacteria (and anaerobic export of succinate) is achieved under aerobic and anaerobic conditions by different sets of secondary transporters. Expression of the genes for C4-dicarboxylate metabolism is induced in the presence of external C4-dicarboxylates by the membrane-bound DcuS-DcuR two-component system. Noncommon C4-dicarboxylates like l-tartrate or D-malate are perceived by cytoplasmic one-component sensors/transcriptional regulators. This article describes the pathways of aerobic and anaerobic C4-dicarboxylate metabolism and their regulation. The citric acid cycle, fumarate respiration, and fumarate reductase are covered in other articles and discussed here only in the context of C4-dicarboxylate metabolism. Recent aspects of C4-dicarboxylate metabolism like transport, sensing, and regulation will be treated in more detail. This article is an updated version of an article published in 2004 in EcoSal Plus. The update includes new literature, but, in particular, the sections on the metabolism of noncommon C4-dicarboxylates and their regulation, on the DcuS-DcuR regulatory system, and on succinate production by engineered E. coli are largely revised or new.

Environmental citrate or malonate is degraded by a variety of aerobic or anaerobic bacteria. For selected examples, the genes encoding the specific enzymes of the degradation pathway are described together with the encoded proteins and their catalytic mechanisms. Aerobic bacteria degrade citrate readily by the basic enzyme equipment of the cell if a specific transporter for citrate is available. Anaerobic degradation of citrate in Klebsiella pneumoniae requires the so-called substrate activation module to convert citrate into its thioester with the phosphoribosyl dephospho-CoA prosthetic group of citrate lyase. The citryl thioester is subsequently cleaved into oxaloacetate and the acetyl thioester, from which a new citryl thioester is formed as the turnover continues. The degradation of malonate likewise includes a substrate activation module with a phosphoribosyl dephospho-CoA prosthetic group. The machinery gets ready for turnover after forming the acetyl thioester with the prosthetic group. The acetyl residue is then exchanged by a malonyl residue, which is easily decarboxylated with the regeneration of the acetyl thioester. This equipment suffices for aerobic growth on malonate, since ATP is produced via the oxidation of acetate. Anaerobic growth on citrate or malonate, however, depends on additional enzymes of a so-called energy conservation module. This allows the conversion of decarboxylation energy into an electrochemical gradient of Na+ ions. In citrate-fermenting K. pneumoniae, the Na+ gradient is formed by the oxaloacetate decarboxylase and mainly used to drive the active transport of citrate into the cell. To use this energy source for this purpose is possible, since ATP is generated by substrate phosphorylation in the well-known sequence from pyruvate to acetate. In the malonate-fermenting bacterium Malonomonas rubra, however, no reactions for substrate level phosphorylation are available and the Na+ gradient formed in the malonate decarboxylation reaction must therefore be used as the driving force for ATP synthesis.

This review considers the pathways for the degradation of amino acids and a few related compounds (agmatine, putrescine, ornithine, and aminobutyrate), along with their functions and regulation. Nitrogen limitation and an acidic environment are two physiological cues that regulate expression of several amino acid catabolic genes. The review considers Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella species. The latter is included because the pathways in Klebsiella species have often been thoroughly characterized and also because of interesting differences in pathway regulation. These organisms can essentially degrade all the protein amino acids, except for the three branched-chain amino acids. E. coli, Salmonella enterica serovar Typhimurium, and Klebsiella aerogenes can assimilate nitrogen from D- and L-alanine, arginine, asparagine, aspartate, glutamate, glutamine, glycine, proline, and D- and L-serine. There are species differences in the utilization of agmatine, citrulline, cysteine, histidine, the aromatic amino acids, and polyamines (putrescine and spermidine). Regardless of the pathway of glutamate synthesis, nitrogen source catabolism must generate ammonia for glutamine synthesis. Loss of glutamate synthase (glutamineoxoglutarate amidotransferase, or GOGAT) prevents utilization of many organic nitrogen sources. Mutations that create or increase a requirement for ammonia also prevent utilization of most organic nitrogen sources.

Central Metabolism

Central metabolism of carbohydrates uses the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways. This review reviews the biological roles of the enzymes and genes of these three pathways of E. coli. Glucose, pentoses, and gluconate are primarily discussed as the initial substrates of the three pathways, respectively. The genetic and allosteric regulatory mechanisms of glycolysis and the factors that affect metabolic flux through the pathways are considered here. Despite the fact that a lot of information on each of the reaction steps has been accumulated over the years for E. coli, surprisingly little quantitative information has been integrated to analyze glycolysis as a system. Therefore, the review presents a detailed description of each of the catalytic steps by a systemic approach. It considers both structural and kinetic aspects. Models that include kinetic information of the reaction steps will always contain the reaction stoichiometry and therefore follow the structural constraints, but in addition to these also kinetic rate laws must be fulfilled. The kinetic information obtained on isolated enzymes can be integrated using computer models to simulate behavior of the reaction network formed by these enzymes. Successful examples of such approaches are the modeling of glycolysis in S. cerevisiae, the parasite Trypanosoma brucei, and the red blood cell. With the rapid developments in the field of Systems Biology many new methods have been and will be developed, for experimental and theoretical approaches, and the authors expect that these will be applied to E. coli glycolysis in the near future.

The tricarboxylic acid (TCA) cycle plays two essential roles in metabolism. First, under aerobic conditions the cycle is responsible for the total oxidation of acetyl-CoA that is derived mainly from the pyruvate produced by glycolysis. Second, TCA cycle intermediates are required in the biosynthesis of several amino acids. Although the TCA cycle has long been considered a “housekeeping” pathway in Escherichia coli and Salmonella enterica, the pathway is highly regulated at the transcriptional level. Much of this control is exerted in response to respiratory conditions. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although a few loose ends remain. The realization that a “shadow” TCA cycle exists that proceeds through methylcitrate has cleared up prior ambiguities. The glyoxylate bypass has long been known to be essential for growth on carbon sources such as acetate or fatty acids because this pathway allowsnet conversion of acetyl-CoA to metabolic intermediates. Strains lacking this pathway fail to grow on these carbon sources, since acetate carbon entering the TCA cycle is quantitatively lost as CO2 resulting in the lack of a means to replenish the dicarboxylic acids consumed in amino acid biosynthesis. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although the identity of the small molecule ligand that modulates transcriptional control of the glyoxylate cycle genes by binding to the IclR repressor remains unknown. The activity of the cycle is also exerted at the enzyme level by the reversible phosphorylation of the TCA cycle enzyme isocitrate dehydrogenase catalyzed by a specific kinase/phosphatase to allow isocitratelyase to compete for isocitrate and cleave this intermediate to glyoxylate and succinate.

Pyruvate and acetyl-CoA form the backbone of central metabolism. The nonoxidative cleavage of pyruvate to acetyl-CoA and formate by the glycyl radical enzyme pyruvate formate lyase is one of the signature reactions of mixed-acid fermentation in enterobacteria. Under these conditions, formic acid accounts for up to one-third of the carbon derived from glucose. The further metabolism of acetyl-CoA to acetate via acetyl-phosphate catalyzed by phosphotransacetylase and acetate kinase is an exemplar of substrate-level phosphorylation. Acetyl-CoA can also be used as an acceptor of the reducing equivalents generated during glycolysis, whereby ethanol is formed by the polymeric acetaldehyde/alcohol dehydrogenase (AdhE) enzyme. The metabolism of acetyl-CoA via either the acetate or the ethanol branches is governed by the cellular demand for ATP and the necessity to reoxidize NADH. Consequently, in the absence of an electron acceptor mutants lacking either branch of acetyl-CoA metabolism fail to cleave pyruvate, despite the presence of PFL, and instead reduce it to D-lactate by the D-lactate dehydrogenase. The conversion of PFL to the active, radical-bearing species is controlled by a radical-SAM enzyme, PFL-activase. All of these reactions are regulated in response to the prevalent cellular NADH:NAD+ ratio. In contrast to Escherichia coli and Salmonella species, some genera of enterobacteria, e.g., Klebsiella and Enterobacter, produce the more neutral product 2,3-butanediol and considerable amounts of CO2 as fermentation products. In these bacteria, two molecules of pyruvate are converted to α-acetolactate (AL) by α-acetolactate synthase (ALS). AL is then decarboxylated and subsequently reduced to the product 2,3-butandiol.

Numerous recent developments in the biochemistry, molecular biology, and physiology of formate and H2 metabolism and of the [NiFe]-hydrogenase (Hyd) cofactor biosynthetic machinery are highlighted. Formate export and import by the aquaporin-like pentameric formate channel FocA is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate. Formate is disproportionated by the reversible formate hydrogenlyase (FHL) complex, which has been isolated, allowing biochemical dissection of evolutionary parallels with complex I of the respiratory chain. A recently identified sulfido-ligand attached to Mo in the active site of formate dehydrogenases led to the proposal of a modified catalytic mechanism. Structural analysis of the homologous, H2-oxidizing Hyd-1 and Hyd-5 identified a novel proximal [4Fe-3S] cluster in the small subunit involved in conferring oxygen tolerance to the enzymes. Synthesis of Salmonella Typhimurium Hyd-5 occurs aerobically, which is novel for an enterobacterial Hyd. The O2-sensitive Hyd-2 enzyme has been shown to be reversible: it presumably acts as a conformational proton pump in the H2-oxidizing mode and is capable of coupling reverse electron transport to drive H2 release. The structural characterization of all the Hyp maturation proteins has given new impulse to studies on the biosynthesis of the Fe(CN)2CO moiety of the [NiFe] cofactor. It is synthesized on a Hyp-scaffold complex, mainly comprising HypC and HypD, before insertion into the apo-large subunit. Finally, clear evidence now exists indicating that Escherichia coli can mature Hyd enzymes differentially, depending on metal ion availability and the prevailing metabolic state. Notably, Hyd-3 of the FHL complex takes precedence over the H2-oxidizing enzymes.

Biosynthesis

By the mid1960s, the pioneering work of Umbarger and Gerhart and Pardee had shown us that carbon flow through a biosynthetic pathway was controlled by allosteric inhibition of the first enzyme of the pathway by its end product; and, studies of the lac operon by Jacob and Monod had established that genes were controlled by an operator-repressor mechanism. During the intervening forty-plus years, knowledge and technologies have continued to explode in unanticipated ways. Today, we understand in great detail the molecular mechanisms of the many levels of metabolic and genetic regulation that control carbon flow through the amino acid biosynthetic pathways. Traditional experimental approaches are not sufficient for the integration and reconstruction of complex biological systems using data mostly generated by high-throughput experiments. Only with computational methods and adequate modeling tools will we be able to reconstruct and query these large and complicated systems. Due to complicated enzyme reaction mechanisms and the frequent lack of rate constant measurements needed for solving differential equations, most investigators have turned their attention to the development of abstract, top-down modeling tools. For example, Palsson and colleagues have used metabolic flux balance analysis (FBA) methods to simulate steady-state metabolite flux through E. coli pathways representing hundreds of enzyme steps. Recently, Yang et al. have developed a bottom-up, enzyme mechanism modeling language, kMech (kinetic mechanism), for the mathematical simulation of metabolic pathways.

About 50 years ago, research on the biological function of the element selenium was initiated by the report of J. Pinsent that generation of formate dehydrogenase activity by Escherichia coli requires the presence of both selenite and molybdate in the growth medium. In nature, selenium is predominantly associated with sulfur minerals, the Se/S ratios of which vary widely depending on the geological formation. Because of the chemical similarity between the two elements, selenium can intrude into the sulfur pathway at high Se/S ratios and can be statistically incorporated into polypeptides. The central macromolecule for the synthesis and incorporation of selenocysteine is a specialized tRNA, designated tRNASec. It is the product of the selC (previously fdhC) gene. tRNASec fulfils a multitude of functions, which are based on its unique structural properties, compared to canonical elongator RNAs. tRNASec possesses the discriminator base G73 and the identity elements of serine-specific tRNA isoacceptors. The conversion of seryl-tRNASec into selenocysteyl-tRNASec is catalyzed by selenocysteine synthase, the product of the selA gene (previously the fdhA locus, which was later shown to harbor two genes, selA and selB). The crucial element for the regulation is a putative secondary structure at the 5′ end of the untranslated region of the selAB mRNA. The generation and analysis of transcriptional and translational reporter gene fusions of selA and selB yield an expression pattern identical to that obtained by measuring the actual amounts of SelA and SelB proteins.

Selenocysteine is a naturally occurring analog of cysteine in which the sulfur atom of the latter is replaced with selenium. This seleno-amino acid occurs as a specific component of various selenoproteins and selenium-dependent enzymes. Incorporation of selenocysteine into these proteins occurs cotranslationally as directed by the UGA codon. For this process, a special tRNA having an anticodon complimentary to UGA, tRNASec, is utilized. In Escherichia coli and related bacteria, this tRNA first is amino acylated with serine, and the seryl-tRNASec is converted to selenocysteyl-tRNASec. The specific incorporation of selenocysteine into proteins directed by the UGA codon depends on the synthesis of selenocysteyl-tRNASec. Included in the selenium delivery protein category are rhodaneses that mobilize selenium from inorganic sources and NIFS-like proteins that liberate elemental selenium from selenocysteine. The NIFS protein from Azotobacter vinelandii was found to serve as an efficient catalyst in vitro for delivery of selenium from free selenocysteine to Escherichia coli selenophosphate synthetase for selenophosphate formation. The widespread distribution of selenocysteine lyase in numerous bacterial species was reported and the bacterial enzymes, like the pig liver enzyme, required pyridoxal phosphate as cofactor. Three NIFS-like genes were isolated from E. coli by Esaki and coworkers and the expressed gene products were isolated and characterized. One of these NIFS-like proteins also exhibited a high preference for selenocysteine over cysteine. M. vannielii, an anaerobic methane-producing organism, that grows in a mineral medium containing formate as sole organic carbon source, synthesizes several specific selenoenzymes required for growth and energy production under these conditions.

Selenophosphate synthetase, the selD gene product from Escherichia coli, is one of the enzymes required for the synthesis and specific insertion of selenocysteine into proteins directed by the TGA codon. Selenophosphate synthetases have been isolated from or are thought to be present in most organisms; however, the best characterized selenophosphate synthetase is from E. coli, in which both in vivo and in vitro studies have been performed. Leinfelder and coworkers showed that an E. coli mutant lacking an intact selD gene fails to incorporate Se into both the selenocysteine-containing enzyme formate dehydrogenase (FDH) and tRNA species that normally contain 2-selenouridine residues at the wobble position. Thus, this study strongly implicated selenophosphate as playing a major role in E. coli selenium metabolic pathways. The selenophosphate synthetase reaction requires some form of reduced selenium such as hydrogen selenide (HSe-) and ATP as substrates to generate a stoichiometric amount of SePO3, AMP, and orthophosphate. Studies of selenophosphate inhibition have provided further insight into the mechanism of selenophosphate synthetase. An assay by which AMP formation is measured in the absence of selenide showed that selenophosphate synthetase catalyzes hydrolysis of ATP to AMP and two orthophosphates in an uncoupled reaction. The sequencing of selenophosphate synthetase genes from various organisms reveals several conserved regions in the gene product. Recent investigations into the mechanism of selenophosphate synthetase have revealed a property of selenophosphate synthetase not previously observed. In samples of purified selenophosphate synthetase, an unusual optical absorption spectrum is seen.

The biosynthesis of serine, glycine, and one-carbon (C1) units constitutes a major metabolic pathway in Escherichia coli and Salmonella enterica serovar Typhimurium. C1 units derived from serine and glycine are used in the synthesis of purines, histidine, thymine, pantothenate, and methionine and in the formylation of the aminoacylated initiator fMet-TRNAfMet used to start translation in E. coli and serovar Typhimurium. The need for serine, glycine, and C1 units in many cellular functions makes it necessary for the genes encoding enzymes for their synthesis to be carefully regulated to meet the changing demands of the cell for these intermediates. This review discusses the regulation of the following genes: serA, serB, and serC; gly gene; gcvTHP operon; lpdA; gcvA and gcvR; and gcvB genes. Threonine utilization (the Tut cycle) constitutes a secondary pathway for serine and glycine biosynthesis. L-Serine inhibits the growth of E. coli cells in GM medium, and isoleucine releases this growth inhibition. The E. coli glycine transport system (Cyc) has been shown to transport glycine, D-alanine, D-serine, and the antibiotic D-cycloserine. Transport systems often play roles in the regulation of gene expression, by transporting effector molecules into the cell, where they are sensed by soluble or membrane-bound regulatory proteins.

Glutamate, aspartate, asparagine, L-alanine, and D-alanine are derived from intermediates of central metabolism, mostly the citric acid cycle, in one or two steps. While the pathways are short, the importance and complexity of the functions of these amino acids befit their proximity to central metabolism. Inorganic nitrogen (ammonia) is assimilated into glutamate, which is the major intracellular nitrogen donor. Glutamate is a precursor for arginine, glutamine, proline, and the polyamines. Glutamate degradation is also important for survival in acidic environments, and changes in glutamate concentration accompany changes in osmolarity. Aspartate is a precursor for asparagine, isoleucine, methionine, lysine, threonine, pyrimidines, NAD, and pantothenate; a nitrogen donor for arginine and purine synthesis; and an important metabolic effector controlling the interconversion of C3 and C4 intermediates and the activity of the DcuS-DcuR two-component system. Finally, L- and D-alanine are components of the peptide of peptidoglycan, and L-alanine is an effector of the leucine responsive regulatory protein and an inhibitor of glutamine synthetase (GS). This review summarizes the genes and enzymes of glutamate, aspartate, asparagine, L-alanine, and D-alanine synthesis and the regulators and environmental factors that control the expression of these genes. Glutamate dehydrogenase (GDH) deficient strains of E. coli, K. aerogenes, and S. enterica serovar Typhimurium grow normally in glucose containing (energy-rich) minimal medium but are at a competitive disadvantage in energy limited medium. Glutamate, aspartate, asparagine, L-alanine, and D-alanine have multiple transport systems.

Proline was among the last biosynthetic precursors to have its biosynthetic pathway unraveled. This review recapitulates the findings on the biosynthesis and transport of proline. Glutamyl kinase (GK) catalyzes the ATP-dependent phosphorylation of L-glutamic acid. Purification of γ-GK from Escherichia coli was facilitated by the expression of the proB and proA genes from a high-copy-number plasmid and the development of a specific coupled assay based on the NADPH-dependent reduction of GP by γ-glutamyl phosphate reductase (GPR). GPR catalyzes the NADPH-dependent reduction of GP to GSA. Site directed mutagenesis was used to identify residues that constitute the active site of E. coli GK. This analysis indicated that there is an overlap between the binding sites for glutamate and the allosteric inhibitor proline, suggesting that proline competes with the binding of glutamate. The review also summarizes the genes involved in the metabolism of proline in E. coli and Salmonella. Among the completed genomic sequences of Enterobacteriaceae, genes specifying all three proline biosynthetic enzymes can be discerned in E. coli, Shigella, Salmonella enterica, Serratia marcescens, Erwinia carotovora, Yersinia, Photorhabdus luminescens, and Sodalis glossinidius strain morsitans. The intracellular proline concentration increases with increasing external osmolality in proline-overproducing mutants. This apparent osmotic regulation of proline accumulation in the overproducing strains may be the result of increased retention or recapture of proline, achieved by osmotic stimulation of the ProP or ProU proline transport systems. A number of proline analogs can be incorporated into proteins in vivo or in vitro.

This review focuses on more recent studies concerning the systems biology of branched-chain amino acid biosynthesis, that is, the pathway-specific and global metabolic and genetic regulatory networks that enable the cell to adjust branched-chain amino acid synthesis rates to changing nutritional and environmental conditions. It begins with an overview of the enzymatic steps and metabolic regulatory mechanisms of the pathways and descriptions of the genetic regulatory mechanisms of the individual operons of the isoleucine-leucine-valine (ilv) regulon. This is followed by more-detailed discussions of recent evidence that global control mechanisms that coordinate the expression of the operons of this regulon with one another and the growth conditions of the cell are mediated by changes in DNA supercoiling that occur in response to changes in cellular energy charge levels that, in turn, are modulated by nutrient and environmental signals. Since the parallel pathways for isoleucine and valine biosynthesis are catalyzed by a single set of enzymes, and because the AHAS-catalyzed reaction is the first step specific for valine biosynthesis but the second step of isoleucine biosynthesis, valine inhibition of a single enzyme for this enzymatic step might compromise the cell for isoleucine or result in the accumulation of toxic intermediates. The operon-specific regulatory mechanisms of the operons of the ilv regulon are discussed in the review followed by a consideration and brief review of global regulatory proteins such as integration host factor (IHF), Lrp, and CAP (CRP) that affect the expression of these operons.

Detailed studies of the glutamine synthetase (GS) in Escherichia coli and other bacteria have shown that the activity of this enzyme is regulated by at least five different mechanisms: (i) cumulative feedback inhibition by multiple end products of glutamine metabolism, (ii) interconversion between taut and relaxed protein configurations in response to binding and dissociation of divalent cations at one of its two metal binding sites, (iii) dynamic interconversion of the enzyme between covalently modified (adenylylated) and unmodified forms by a novel bicyclic cascade system, (iv) repression and derepression of glutamine synthetase formation by cyclic phosphorylation and dephosphorylation of an RNA factor that governs transcription activities, and (v) regulation of glutamine synthetase turnover by the coupling of site specific metal ion-catalyzed oxidation with proteolytic degradation of the enzyme. Glutamine synthetase activity in E. coli is subject to inhibition by seven different end products of glutamine metabolism, namely, by tryptophan, histidine, carbamyl-phosphate, CTP, AMP, glucose-6-phosphate, and NAD+, and also by serine, alanine, and glycine. The cascade theory predicts that the steady-state level of glutamine synthetase adenylylation and therefore its catalytic activity is determined by the combined effects of all metabolites that affect the kinetic parameters of one or more of the enzymes in the cascade. Furthermore, under conditions where the supplies of ATP and glutamate are not limiting and the production of glutamine exceeds the demand, GS is no longer needed, then it will be converted to the catalytically inactive adenylylated form that is not under protection of ATP and glutamate.

This review focuses on the steps unique to methionine biosynthesis, namely the conversion of homoserine to methionine. The past decade has provided a wealth of information concerning the details of methionine metabolism and the review focuses on providing a comprehensive overview of the field, emphasizing more recent findings. Details of methionine biosynthesis are addressed along with key cellular aspects, including regulation, uptake, utilization, AdoMet, the methyl cycle, and growing evidence that inhibition of methionine biosynthesis occurs under stressful cellular conditions. The first unique step in methionine biosynthesis is catalyzed by the metA gene product, homoserine transsuccinylase (HTS, or homoserine O-succinyltransferase). Recent experiments suggest that transcription of these genes is indeed regulated by MetJ, although the repressor-binding sites have not yet been verified. Methionine also serves as the precursor of S-adenosylmethionine, which is an essential molecule employed in numerous biological processes. S-adenosylhomocysteine is produced as a consequence of the numerous AdoMet-dependent methyl transfer reactions that occur within the cell. In E. coli and Salmonella, this molecule is recycled in two discrete steps to complete the methyl cycle. Cultures challenged by oxidative stress appear to experience a growth limitation that depends on methionine levels. E. coli that are deficient for the manganese and iron superoxide dismutases (the sodA and sodB gene products, respectively) require the addition of methionine or cysteine for aerobic growth. Modulation of methionine levels in response to stressful conditions further increases the complexity of its regulation.

This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon.

The biosynthesis of histidine in Escherichia coli and Salmonella typhimurium has been an important model system for the study of relationships between the flow of intermediates through a biosynthetic pathway and the control of the genes encoding the enzymes that catalyze the steps in a pathway. This article provides a comprehensive review of the histidine biosynthetic pathway and enzymes, including regulation of the flow of intermediates through the pathway and mechanisms that regulate the amounts of the histidine biosynthetic enzymes. In addition, this article reviews the structure and regulation of the histidine (his) biosynthetic operon, including transcript processing, Rho-factor-dependent “classical” polarity, and the current model of his operon attenuation control. Emphasis is placed on areas of recent progress. Notably, most of the enzymes that catalyze histidine biosynthesis have recently been crystallized, and their structures have been determined. Many of the histidine biosynthetic intermediates are unstable, and the histidine biosynthetic enzymes catalyze some chemically unusual reactions. Therefore, these studies have led to considerable mechanistic insight into the pathway itself and have provided deep biochemical understanding of several fundamental processes, such as feedback control, allosteric interactions, and metabolite channeling. Considerable recent progress has also been made on aspects of his operon regulation, including the mechanism of pp(p)Gpp stimulation of his operon transcription, the molecular basis for transcriptional pausing by RNA polymerase, and pathway evolution. The progress in these areas will continue as sophisticated new genomic, metabolomic, proteomic, and structural approaches converge in studies of the histidine biosynthetic pathway and mechanisms of control of his biosynthetic genes in other bacterial species.

Early investigations on arginine biosynthesis brought to light basic features of metabolic regulation. The most significant advances of the last 10 to 15 years concern the arginine repressor, its structure and mode of action in both E. coli and Salmonella typhimurium, the sequence analysis of all arg structural genes in E. coli and Salmonella typhimurium, the resulting evolutionary inferences, and the dual regulation of the carAB operon. This review provides an overall picture of the pathways, their interconnections, the regulatory circuits involved, and the resulting interferences between arginine and polyamine biosynthesis. Carbamoylphosphate is a precursor common to arginine and the pyrimidines. In both Escherichia coli and Salmonella enterica serovar Typhimurium, it is produced by a single synthetase, carbamoylphosphate synthetase (CPSase), with glutamine as the physiological amino group donor. This situation contrasts with the existence of separate enzymes specific for arginine and pyrimidine biosynthesis in Bacillus subtilis and fungi. Polyamine biosynthesis has been particularly well studied in E. coli, and the cognate genes have been identified in the Salmonella genome as well, including those involved in transport functions. The review summarizes what is known about the enzymes involved in the arginine pathway of E. coli and S. enterica serovar Typhimurium; homologous genes were identified in both organisms, except argF (encoding a supplementary OTCase), which is lacking in Salmonella. Several examples of putative enzyme recruitment (homologous enzymes performing analogous functions) are also presented.

The synthesis of L-cysteine from inorganic sulfur is the predominant mechanism by which reduced sulfur is incorporated into organic compounds. L-cysteineis used for protein and glutathione synthesis and serves as the primary source of reduced sulfur in L-methionine, lipoic acid, thiamin, coenzyme A (CoA), molybdopterin, and other organic molecules. Sulfate and thiosulfate uptake in E. coli and serovar Typhimurium are achieved through a single periplasmic transport system that utilizes two different but similar periplasmic binding proteins. Kinetic studies indicate that selenate and selenite share a single transporter with sulfate, but molybdate also has a separate transport system. During aerobic growth, the reduction of sulfite to sulfide is catalyzed by NADPH-sulfite reductase (SiR), and serovar Typhimurium mutants lacking this enzyme accumulate sulfite from sulfate, implying that sulfite is a normal intermediate in assimilatory sulfate reduction. L-Cysteine biosynthesis in serovar Typhimurium and E. coli ceases almost entirely when cells are grown on L-cysteine or L-cystine, owing to a combination of end product inhibition of serine transacetylase by L-cysteine and a gene regulatory system known as the cysteine regulon, wherein genes for sulfate assimilation and alkanesulfonate utilization are expressed only when sulfur is limiting. In vitro studies with the cysJIH, cysK, and cysP promoters have confirmed that they are inefficient at forming transcription initiation complexes without CysB and N-acetyl-L-serine. Activation of the tauA and ssuE promoters requires Cbl. It has been proposed that the three serovar Typhimurium anaerobic reductases for sulfite, thiosulfate, and tetrathionate may function primarily in anaerobic respiration.

We review literature on the metabolism of ribo- and deoxyribonucleotides, nucleosides, and nucleobases in Escherichia coli and Salmonella,including biosynthesis, degradation, interconversion, and transport. Emphasis is placed on enzymology and regulation of the pathways, at both the level of gene expression and the control of enzyme activity. The paper begins with an overview of the reactions that form and break the N-glycosyl bond, which binds the nucleobase to the ribosyl moiety in nucleotides and nucleosides, and the enzymes involved in the interconversion of the different phosphorylated states of the nucleotides. Next, the de novo pathways for purine and pyrimidine nucleotide biosynthesis are discussed in detail.Finally, the conversion of nucleosides and nucleobases to nucleotides, i.e.,the salvage reactions, are described. The formation of deoxyribonucleotides is discussed, with emphasis on ribonucleotidereductase and pathways involved in fomation of dUMP. At the end, we discuss transport systems for nucleosides and nucleobases and also pathways for breakdown of the nucleobases.

Glycogen accumulation occurs in Escherichia coli and Salmonella enterica serovar Typhimurium as well as in many other bacteria. Glycogen will be formed when there is an excess of carbon under conditions in which growth is limited because of the lack of a growth nutrient, e.g., a nitrogen source. This review describes the enzymatic reactions involved in glycogen synthesis and the allosteric regulation of the first enzyme, ADP-glucose pyrophosphorylase. The properties of the enzymes involved in glycogen synthesis, ADP-glucose pyrophosphorylase, glycogen synthase, and branching enzyme are also characterized. The data describing the genetic regulation of the glycogen synthesis are also presented. An alternate pathway for glycogen synthesis in mycobacteria is also described.

The pathways in Escherichia coli and (largely by analogy) S. enterica remain the paradigm of bacterial lipid synthetic pathways, although recently considerable diversity among bacteria in the specific areas of lipid synthesis has been demonstrated. The structural biology of the fatty acid synthetic proteins is essentially complete. However, the membrane-bound enzymes of phospholipid synthesis remain recalcitrant to structural analyses. Recent advances in genetic technology have allowed the essentialgenes of lipid synthesis to be tested with rigor, and as expected most genes are essential under standard growth conditions. Conditionally lethal mutants are available in numerous genes, which facilitates physiological analyses. The array of genetic constructs facilitates analysis of the functions of genes from other organisms. Advances in mass spectroscopy have allowed very accurate and detailed analyses of lipid compositions as well as detection of the interactions of lipid biosynthetic proteins with one another and with proteins outside the lipid pathway. The combination of these advances has resulted in use of E. coli and S. enterica for discovery of new antimicrobials targeted to lipid synthesis and in deciphering the molecular actions of known antimicrobials. Finally,roles for bacterial fatty acids other than as membrane lipid structural components have been uncovered. For example, fatty acid synthesis plays major roles in the synthesis of the essential enzyme cofactors, biotin and lipoic acid. Although other roles for bacterial fatty acids, such as synthesis of acyl-homoserine quorum-sensing molecules, are not native to E. coli introduction of the relevant gene(s) synthesis of these foreign molecules readily proceeds and the sophisticated tools available can used to decipher the mechanisms of synthesis of these molecules.

Coenzymes, Cofactors and Prosthetic Groups

The biosynthesis of riboflavin requires 1 equivalent of GTP and 2 equivalents of ribulose phosphate. The first committed reactions of the convergent pathway are catalyzed by GTP hydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase. The initial reaction steps afford 5-amino-6-ribitylaminopyrimidine 5′-phosphate, which needs to be dephosphorylated by a hitherto elusive hydrolase. The dephosphorylated pyrimidine is condensed with the carbohydrate precursor, 3,4-dihydroxy-2-butanone 4-phosphate. The resulting 6,7-dimethyl-8-ribityllumazine affords riboflavin by a mechanistically unique dismutation, i.e., by formation of a pentacyclic dimer that is subsequently fragmented.

Escherichia coli and Salmonella contain the naphthoquinones menaquinone (MK; vitamin K2) and demethylmenaquinone and the benzoquinone ubiquinone (coenzyme Q; Q). Both quinones are derived from the shikimate pathway, which has been called a "metabolic tree with many branches." There are two different pathways for the biosynthesis of the naphthoquinones. The vast majority of prokaryotes, including E. coli and Salmonella, and the plants use the o-succinylbenzoate pathway, while a minority uses the futalosine pathway. The quinone nucleus of Q is derived directly from chorismate, while that of MK is derived from chorismate via isochorismate. The prenyl side chains of both quinones are from isopentenyl diphosphate formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway and the methyl groups are from S-adenosylmethionine. In addition, MK biosynthesis requires 2-ketoglutarate and cofactors ATP, coenzyme A, and thiamine pyrophosphate. Despite the fact that both quinones originate from the shikimate pathway, there are important differences in their biosyntheses. The prenyl side chain in MK biosynthesis is introduced at the penultimate step, accompanied by decarboxylation, whereas in Q biosynthesis it is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to prenylation are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis, the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. Despite the fact that Q and MK biosyntheses diverge at chorismate, the C-methylations in both pathways are carried out by the same methyltransferase.

Pantothenate is vitamin B5 and is the key precursor for the biosynthesis of coenzyme A (CoA), a universal and essential cofactor involved in a myriad of metabolic reactions, including the synthesis of phospholipids, the synthesis and degradation of fatty acids, and the operation of the tricarboxylic acid cycle. CoA is also the only source of the phosphopantetheine prosthetic group for enzymes that shuttle intermediates between the active sites of enzymes involved in fatty acid, nonribosomal peptide, and polyketide synthesis. Pantothenate can be synthesized de novo and/or transported into the cell through a pantothenatepermease. Pantothenate uptake is essential for those organisms that lack the genes to synthesize this vitamin. The intracellular levels of CoA are controlled by the balance between synthesis and degradation. In particular, CoA is assembled in five enzymatic steps, starting from the phosphorylation of pantothenate to phosphopantothenatecatalyzed by pantothenate kinase, the product of the coaA gene. In some bacteria, the production of phosphopantothenate by pantothenate kinase is the rate limiting and most regulated step in the biosynthetic pathway. CoA synthesis additionally networks with other vitamin-associated pathways, such as thiamine and folic acid.

Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.

Many microorganisms and plants possess the ability to synthesize folic acid derivatives de novo, initially forming dihydrofolate. All the folic acid derivatives that serve as recipients and donors of one-carbon units are derivatives of tetrahydrofolate, which is formed from dihydrofolate by an NADPH-dependent reduction catalyzed by dihydrofolate reductase (FolA). This review discusses the biosynthesis of dihydrofolate monoglutamate, its reduction to tetrahydrofolate monoglutamate, and the addition of glutamyl residues to form folylpolyglutamates. Escherichia coli and Salmonella, like many microorganisms that can synthesize folate de novo, appear to lack the ability to transport folate into the cell and are thus highly susceptible to inhibitors of folate biosynthesis. The review includes a brief discussion of the inhibition of folate biosynthesis by sulfa drugs. The folate biosynthetic pathway can be divided into two sections. First, the aromatic precursor chorismate is converted to paminobenzoic acid (PABA) by the action of three proteins. Second, the pteridine portion of folate is made from GTP and coupled to PABA to generate dihydropteroate, and the bifunctional protein specified by folC, dihydrofolate synthetase, or folylpolyglutamate synthetase, adds the initial glutamate molecule to form dihydrofolate (H2PteGlu1, or dihydropteroylmonoglutamate). Bacteriophage T4 infection of E. coli has been shown to cause alterations in the metabolism of folate derivatives. Infection is associated with an increase in the chain lengths in folylpolyglutamates and particularly the accumulation of hexaglutamate derivatives.

The biosynthesis of thiamin pyrophosphate (TPP) in prokaryotes, as represented by the Escherichia coli and the Bacillus subtilis pathways, is summarized in this review. The thiazole heterocycle is formed by the convergence of three separate pathways. First, the condensation of glyceraldehyde 3-phosphate and pyruvate, catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), gives 1-deoxy-D-xylulose 5-phosphate (DXP). Next, the sulfur carrier protein ThiS-COO- is converted to its carboxyterminal thiocarboxylate in reactions catalyzed by ThiF, ThiI, and NifS (ThiF and IscS in B. subtilis). Finally, tyrosine (glycine in B. subtilis) is converted to dehydroglycine by ThiH (ThiO in B. subtilis). Thiazole synthase (ThiG) catalyzes the complex condensation of ThiS-COSH, dehydroglycine, and DXP to give a thiazole tautomer, which is then aromatized to carboxythiazole phosphate by TenI (B. subtilis). Hydroxymethyl pyrimidine phosphate (HMP-P) is formed by a complicated rearrangement reaction of 5-aminoimidazole ribotide (AIR) catalyzed by ThiC. ThiD then generates hydroxymethyl pyrimidine pyrophosphate. The coupling of the two heterocycles and decarboxylation, catalyzed by thiamin phosphate synthase (ThiE), gives thiamin phosphate. A final phosphorylation, catalyzed by ThiL, completes the biosynthesis of TPP, the biologically active form of the cofactor. This review reviews the current status of mechanistic and structural studies on the enzymes involved in this pathway. The availability of multiple orthologs of the thiamin biosynthetic enzymes has also greatly facilitated structural studies, and most of the thiamin biosynthetic and salvage enzymes have now been structurally characterized.

This review summarizes research performed over the last 23 years on the genetics, enzyme structures and functions, and regulation of the expression of the genes encoding functions involved in adenosylcobalamin (AdoCbl, or coenzyme B12) biosynthesis. It also discusses the role of coenzyme B12 in the physiology of Salmonella enterica serovar Typhimurium LT2 and Escherichia coli. John Roth's seminal contributions to the field of coenzyme B12 biosynthesis research brought the power of classical and molecular genetic, biochemical, and structural approaches to bear on the extremely challenging problem of dissecting the steps of what has turned out to be one of the most complex biosynthetic pathways known. In E. coli and serovar Typhimurium, uro’gen III represents the first branch point in the pathway, where the routes for cobalamin and siroheme synthesis diverge from that for heme synthesis. The cobalamin biosynthetic pathway in P. denitrificans was the first to be elucidated, but it was soon realized that there are at least two routes for cobalamin biosynthesis, representing aerobic and anaerobic variations. The expression of the AdoCbl biosynthetic operon is complex and is modulated at different levels. At the transcriptional level, a sensor response regulator protein activates the transcription of the operon in response to 1,2-Pdl in the environment. Serovar Typhimurium and E. coli use ethanolamine as a source of carbon, nitrogen, and energy. In addition, and unlike E. coli, serovar Typhimurium can also grow on 1,2-Pdl as the sole source of carbon and energy.

Universal and ubiquitous redox cofactors, nicotinamide adenine dinucleotide (NAD) and its phosphorylated analog (NADP), collectively contribute to approximately 12% of all biochemical reactions included in the metabolic model of Escherichia coli K-12. A homeostasis of the NAD pool faithfully maintained by the cells results from a dynamic balance in a network of NAD biosynthesis, utilization, decomposition, and recycling pathways that is subject to tight regulation at various levels. A brief overview of NAD utilization processes is provided in this review, including some examples of nonredox utilization. The review focuses mostly on those aspects of NAD biogenesis and utilization in E. coli and Salmonella that emerged within the past 12 years. The first pyridine nucleotide cycle (PNC) originally identified in mammalian systems and termed the Preiss-Handler pathway includes a single-step conversion of niacin (Na) to NaMN by nicotinic acid phosphoribosyltransferase (PncB). In E. coli and many other prokaryotes, this enzyme, together with nicotinamide deamidase (PncA), compose the major pathway for utilization of the pyridine ring in the form of amidated (Nm) or deamidated (Na) precursors. The existence of various regulatory mechanisms and checkpoints that control the NAD biosynthetic machinery reflects the importance of maintaining NAD homeostasis in a variety of growth conditions. Among the most important regulatory mechanisms at the level of individual enzymes are a classic feedback inhibition of NadB, the first enzyme of NAD de novo biosynthesis, by NAD and a metabolic regulation of NadK by reduced cofactors.

This review is concerned specifically with the structures and biosynthesis of hemes in E. coli and serovar Typhimurium. However, inasmuch as all tetrapyrroles share a common biosynthetic pathway, much of the material covered here is applicable to tetrapyrrole biosynthesis in other organisms. Conversely, much of the available information about tetrapyrrole biosynthesis has been gained from studies of other organisms, such as plants, algae, cyanobacteria, and anoxygenic phototrophs, which synthesize large quantities of these compounds. This information is applicable to E. coli and serovar Typhimurium. Hemes play important roles as enzyme prosthetic groups in mineral nutrition, redox metabolism, and gas-and redox-modulated signal transduction. The biosynthetic steps from the earliest universal precursor, 5-aminolevulinic acid (ALA), to protoporphyrin IX-based hemes constitute the major, common portion of the pathway, and other steps leading to specific groups of products can be considered branches off the main axis. Porphobilinogen (PBG) synthase (PBGS; also known as ALA dehydratase) catalyzes the asymmetric condensation of two ALA molecules to form PBG, with the release of two molecules of H2O. Protoporphyrinogen IX oxidase (PPX) catalyzes the removal of six electrons from the tetrapyrrole macrocycle to form protoporphyrin IX in the last biosynthetic step that is common to hemes and chlorophylls. Several lines of evidence converge to support a regulatory model in which the cellular level of available or free protoheme controls the rate of heme synthesis at the level of the first step unique to heme synthesis, the formation of GSA by the action of GTR.

Escherichia coli employs several c-type cytochromes, which are found in the periplasm or on the periplasmic side of the cytoplasmic membrane; they are used for respiration under different growth conditions. All E. colic-type cytochromes are multiheme cytochromes; E. coli does not have a monoheme cytochrome c of the kind found in mitochondria. The attachment of heme to cytochromes c occurs in the periplasm, and so the apoprotein must be transported across the cytoplasmic membrane; this step is mediated by the Sec system, which transports unfolded proteins across the membrane. The protein CcmE has been found to bind heme covalently via a single bond and then transfer the heme to apocytochromes. It should be mentioned that far less complex systems for cytochrome c biogenesis exist in other organisms and that enterobacteria do not function as a representative model system for the process in general, although plant mitochondria use the Ccm system found in E. coli. The variety and distribution of cytochromes and their biogenesis systems reflect their significance and centrality in cellular bioenergetics, though the necessity for and origin of the diverse biogenesis systems are enigmatic.

The transition element molybdenum (Mo) is of primordial importance for biological systems, because it is required by enzymes catalyzing key reactions in the global carbon, sulfur, and nitrogen metabolism. To gain biological activity, Mo has to be complexed by a special cofactor. With the exception of bacterial nitrogenase, all Mo-dependent enzymes contain a unique pyranopterin-based cofactor coordinating a Mo atom at their catalytic site. Various types of reactions are catalyzed by Mo-enzymes in prokaryotes including oxygen atom transfer, sulfur or proton transfer, hydroxylation, or even nonredox reactions. Mo-enzymes are widespread in prokaryotes and many of them were likely present in the Last Universal Common Ancestor. To date, more than 50 – mostly bacterial – Mo-enzymes are described in nature. In a few eubacteria and in many archaea, Mo is replaced by tungsten bound to the same unique pyranopterin. How Mo-cofactor is synthesized in bacteria is reviewed as well as the way until its insertion into apo-Mo-enzymes.

This review describes the two main systems, namely the Isc (iron-sulfur cluster) and Suf (sulfur assimilation) systems, utilized by Escherichia coli and Salmonella for the biosynthesis of iron-sulfur (Fe-S) clusters, as well as other proteins presumably participating in this process. In the case of Fe-S cluster biosynthesis, it is assumed that the sulfur atoms from the cysteine desulfurase end up at cysteine residues of the scaffold protein, presumably waiting for iron atoms for cluster assembly. The review discusses the various potential iron donor proteins. For in vitro experiments, in general, ferrous salts are used during the assembly of Fe-S clusters, even though this approach is unlikely to reflect the physiological conditions. The fact that sulfur atoms can be directly transferred from cysteine desulfurases to scaffold proteins supports a mechanism in which the latter bind sulfur atoms first and iron atoms afterwards. In E. coli, fdx gene inactivation results in a reduced growth rate and reduced Fe-S enzyme activities. Interestingly, the SufE structure resembles that of IscU, strengthening the notion that the two proteins share the property of acting as acceptors of sulfur atoms provided by cysteine desulfurases. Several other factors have been suggested to participate in cluster assembly and repair in E. coli and Salmonella. Most of them were identified by their abilities to act as extragenic and/or multicopy suppressors of mutations in Fe-S cluster metabolism, while others possess biochemical properties that are consistent with a role in Fe-S cluster biogenesis.

Domain 4

Protein

Citation: Peter B. Moore. How we got to where we are: the ribosome in the 21st century. doi:10.1128/ecosalplus.2.5.1

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This article is a short, informal history of the ribosome field that begins with the emergence of the field in the 1930s and ends with a description of its state in 2007, the year this essay was written. The growth in our understanding of both the role of the ribosome in protein synthesis and its structure is emphasized. Starting in 2000, the field experienced a massive upheaval as a result of the publication of the first atomic-resolution crystal structures for ribosomes. However, by 2007, the field had recovered sufficiently so that one could begin to understand how it was likely to evolve in its "poststructural" era. For that reason, this essay is about as useful as a short history of the ribosome field today as it was several years ago, when it was written.

Citation: Jack A. Dunkle, Jamie H. D. Cate. An introduction to the structure and function of the ribosome. doi:10.1128/ecosal.2.5.2

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E. coli continues to serve as a key model for the structure and function of the ribosome, structures of ribosome from other organisms and domains of life have also greatly contributed to our knowledge of protein synthesis. Many structural models of the ribosome in a number of steps of the protein synthesis cycle have been solved by cryo-electron microscopy (cryo-EM) and x-ray crystallography. This chapter introduces the structure and dynamics of the ribosome based on these structures and ends with a brief discussion of the many questions that the structures leave unanswered. Protein synthesis is a multistep process, and the structural features of the ribosome along with the large number of cofactors reflect the complexity of translation. Numerous protein factors in addition to the ribosome contribute to translation in bacteria during the steps of initiation, elongation, termination, and recycling. These protein factors make intimate contacts to key regions of the ribosome, and this aspect is discussed in the chapter in light of our present understanding of the structure and function of the ribosome. The intact ribosome contains three binding sites for substrate tRNAs that are termed as the aminoacyl-tRNA site (A site), peptidyl-tRNA site (P site), and exit-tRNA site (E site). These three binding sites span the interface between the 30S and 50S subunits. The central activity of the ribosome is catalysis of peptide bond formation. The region of the ribosome responsible for catalyzing the reaction is called the peptidyl transferase center (PTC).

Protein synthesis involves nearly a third of the total molecules in a typical bacterial cell. Within the cell, protein synthesis is performed by the ribosomes, and research over several decades has investigated ribosomal formation, structure, and function. This review provides an overview of the current understanding of the assembly of the Escherichia coli 30S ribosomal subunit. The E. coli 30S subunit contains one rRNA molecule (16S) and 21 ribosomal proteins (r-proteins; S1 to S21). The formation of functional subunits can occur as a self-assembly process in vitro; i.e., all the information required for the formation of active ribosomes resides in the primary sequences of the r-proteins and rRNAs. In vitro reconstitution of functional 30S subunits is carried out by using a mixture of TP30, individually purified natural or recombinant r-proteins, and natural 16S rRNA. Chemical probing and primer extension analysis have been used extensively to monitor changes in the reactivities of nucleotides in 16S rRNA during the in vitro reconstitution of 30S subunits. The potential roles for r-proteins in 30S subunit assembly were determined by omitting single proteins in reconstitution experiments. The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. Recent developments in understanding the structure of the 30S subunit have led to speculation about roles for some of the r-proteins in assembly. The crystal structures of the 30S subunit (1, 2) and the 70S ribosome (3) reveal details of the r-protein and rRNA interactions.

The bacterial ribosome is a complex macromolecular machine that deciphers the genetic code with remarkable fidelity. During the elongation phase of protein synthesis, the ribosome selects aminoacyl-tRNAs as dictated by the canonical base pairing between the anticodon of the tRNA and the codon of the messenger RNA. The ribosome's participation in tRNA selection is active rather than passive, using conformational changes of conserved bases of 16S rRNA to directly monitor the geometry of codon-anticodon base pairing. The tRNA selection process is divided into an initial selection step and a subsequent proofreading step, with the utilization of two sequential steps increasing the discriminating power of the ribosome far beyond that which could be achieved based on the thermodynamics of codon-anticodon base pairing stability. The accuracy of decoding is impaired by a number of antibiotics and can be either increased or decreased by various mutations in either subunit of the ribosome, in elongation factor Tu, and in tRNA. In this chapter we will review our current understanding of various forces that determine the accuracy of decoding by the bacterial ribosome.

Citation: Antón Vila-Sanjurjo. Modification of the ribosome and the translational machinery during reduced growth due to environmental stress. doi:10.1128/ecosalplus.2.5.6

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Escherichia coli strains normally used under laboratory conditions have been selected for maximum growth rates and require maximum translation efficiency. Recent studies have shed light on the structural and functional changes undergone by the translational machinery in E. coli during heat and cold shock and upon entry into stationary phase. In these situations both the composition and the partitioning of this machinery into the different pools of cellular ribosomes are modified. As a result, the translational capacity of the cell is dramatically altered. This review provides a comprehensive account of these modifications, regardless of whether or not their underlying mechanisms and their effects on cellular physiology are known. Not only is the composition of the ribosome modified upon entry into stationary phase, but the modification of other components of the translational machinery, such as elongation factor Tu (EFTu) and tRNAs, has also been observed. Hibernation-promoting factor (HPF), paralog protein Y (PY), and ribosome modulation factor (RMF) may also be related to the general protection against environmental stress observed in stationary-phase E. coli cells, a role that would not be revealed necessarily by the viability assays. Even for the best-characterized ribosome-associated factors induced under stress (RMF, PY, and initiation factors), we are far from a complete understanding of their modes of action.

Citation: Katherine S. Long, Birte Vester. Antibiotic resistance mechanisms, with an emphasis on those related to the ribosome. doi:10.1128/ecosalplus.2.5.7

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Antibiotic resistance is a fundamental aspect of microbiology, but it is also a phenomenon of vital importance in the treatment of diseases caused by pathogenic microorganisms. A resistance mechanism can involve an inherent trait or the acquisition of a new characteristic through either mutation or horizontal gene transfer. The natural susceptibilities of bacteria to a certain drug vary significantly from one species of bacteria to another and even from one strain to another. Once inside the cell, most antibiotics affect all bacteria similarly. The ribosome is a major site of antibiotic action and is targeted by a large and chemically diverse group of antibiotics. A number of these antibiotics have important applications in human and veterinary medicine in the treatment of bacterial infections. The antibiotic binding sites are clustered at functional centers of the ribosome, such as the decoding center, the peptidyl transferase center, the GTPase center, the peptide exit tunnel, and the subunit interface spanning both subunits on the ribosome. Upon binding, the drugs interfere with the positioning and movement of substrates, products, and ribosomal components that are essential for protein synthesis. Ribosomal antibiotic resistance is due to the alteration of the antibiotic binding sites through either mutation or methylation. Our knowledge of antibiotic resistance mechanisms has increased, in particular due to the elucidation of the detailed structures of antibiotic-ribosome complexes and the components of the efflux systems. A number of mutations and methyltransferases conferring antibiotic resistance have been characterized. These developments are important for understanding and approaching the problems associated with antibiotic resistance, including design of antimicrobials that are impervious to known bacterial resistance mechanisms.

Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e., the attachment of a proteinogenic amino acid on their cognate tRNAs, thereby mediating the correct expression of the genetic code. In addition, some aaRSs acquired other functions beyond this key role in translation. Genomics and X-ray crystallography have revealed great structural diversity in aaRSs (e.g., in oligomery and modularity, in ranking into two distinct groups each subdivided in 3 subgroups, by additional domains appended on the catalytic modules). AaRSs show huge structural plasticity related to function and limited idiosyncrasies that are kingdom or even species specific (e.g., the presence in many Bacteria of non discriminating aaRSs compensating for the absence of one or two specific aaRSs, notably AsnRS and/or GlnRS). Diversity, as well, occurs in the mechanisms of aaRS gene regulation that are not conserved in evolution, notably between distant groups such as Gram-positive and Gram-negative Bacteria. The review focuses on bacterial aaRSs (and their paralogs) and covers their structure, function, regulation, and evolution. Structure/function relationships are emphasized, notably the enzymology of tRNA aminoacylation and the editing mechanisms for correction of activation and charging errors. The huge amount of genomic and structural data that accumulated in last two decades is reviewed, showing how the field moved from essentially reductionist biology towards more global and integrated approaches. Likewise, the alternative functions of aaRSs and those of aaRS paralogs (e.g., during cell wall biogenesis and other metabolic processes in or outside protein synthesis) are reviewed. Since aaRS phylogenies present promiscuous bacterial, archaeal, and eukaryal features, similarities and differences in the properties of aaRSs from the three kingdoms of life are pinpointed throughout the review and distinctive characteristics of bacterium-like synthetases from organelles are outlined.

The modified nucleosides of RNA are chemically altered versions of the standard A, G, U, and C nucleosides. This review reviews the nature and location of the modified nucleosides of Escherichia coli rRNA, the enzymes that form them, and their known and/or putative functional role. There are seven Ψ (pseudouridines) synthases to make the 11 pseudouridines in rRNA. There is disparity in numbers because RluC and RluD each make 3 pseudouridines. Crystal structures have shown that the Ψ synthase domain is a conserved fold found only in all five families of Ψ synthases. The conversion of uridine to Ψ has no precedent in known metabolic reactions. Other enzymes are known to cleave the glycosyl bond but none carry out rotation of the base and rejoining to the ribose while still enzyme bound. Ten methyltransferases (MTs) are needed to make all the methylated nucleosides in 16S RNA, and 14 are needed for 23S RNA. Biochemical studies indicate that the modes of substrate recognition are idiosyncratic for each Ψ synthase since no common mode of recognition has been detected in studies of the seven synthases. Eight of the 24 expected MTs have been identified, and six crystal structures have been determined. Seven of the MTs and five of the structures are class I MTs with the appropriate protein fold plus unique appendages for the Ψ synthases. The remaining MT, RlmB, has the class IV trefoil knot fold.

Transfer RNA (tRNA) from all organisms on this planet contains modified nucleosides, which are derivatives of the four major nucleosides. tRNA from Escherichia coli/Salmonella enterica serovar Typhimurium contains 33 different modified nucleosides, which are all, except one (Queuosine [Q]), synthesized on an oligonucleotide precursor, which by specific enzymes later matures into tRNA. The structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The synthesis of the tRNA-modifying enzymes is not regulated similarly, and it is not coordinated to that of their substrate, the tRNA. The synthesis of some of them (e.g., several methylated derivatives) is catalyzed by one enzyme, which is position and base specific, whereas synthesis of some has a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N 6-cyclicthreonyladenosine [ct6A], and Q). Several of the modified nucleosides are essential for viability (e.g., lysidin, ct6A, 1-methylguanosine), whereas the deficiency of others induces severe growth defects. However, some have no or only a small effect on growth at laboratory conditions. Modified nucleosides that are present in the anticodon loop or stem have a fundamental influence on the efficiency of charging the tRNA, reading cognate codons, and preventing missense and frameshift errors. Those that are present in the body of the tRNA primarily have a stabilizing effect on the tRNA. Thus, the ubiquitous presence of these modified nucleosides plays a pivotal role in the function of the tRNA by their influence on the stability and activity of the tRNA.

Selection of correct start codons on messenger RNAs is a key step required for faithful translation of the genetic message. Such a selection occurs in a complex process, during which a translation-competent ribosome assembles, eventually having in its P site a specialized methionyl-tRNAMet base-paired with the start codon on the mRNA. This chapter summarizes recent advances describing at the molecular level the successive steps involved in the process. Special emphasis is put on the roles of the three initiation factors and of the initiator tRNA, which are crucial for the efficiency and the specificity of the process. In particular, structural analyses concerning complexes containing ribosomal subunits, as well as detailed kinetic studies, have shed new light on the sequence of events leading to faithful initiation of protein synthesis in Bacteria

Proteins that reside partially or completely outside the bacterial cytoplasm require specialized pathways to facilitate their localization. Globular proteins that function in the periplasm must be translocated across the hydrophobic barrier of the inner membrane. While the Sec pathway transports proteins in a predominantly unfolded conformation, the Tat pathway exports folded protein substrates. Protein transport by the Tat machinery is powered solely by the transmembrane proton gradient, and there is no requirement for nucleotide triphosphate hydrolysis. Proteins are targeted to the Tat machinery by N-terminal signal peptides that contain a consensus twin arginine motif. In Escherichia coli and Salmonella there are approximately thirty proteins with twin arginine signal peptides that are transported by the Tat pathway. The majority of these bind complex redox cofactors such as iron sulfur clusters or the molybdopterin cofactor. Here we describe what is known about Tat substrates in E. coli and Salmonella, the function and mechanism of Tat protein export, and how the cofactor insertion step is coordinated to ensure that only correctly assembled substrates are targeted to the Tat machinery.

Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.

The major class of integral proteins found in the outer membrane (OM) of E. coli and Salmonella adopt a β-barrel conformation (OMPs). OMPs are synthesized in the cytoplasm with a typical signal sequence at the amino terminus, which directs them to the secretion machinery (SecYEG) located in the inner membrane for translocation to the periplasm. Chaperones such as SurA, or DegP and Skp, escort these proteins across the aqueous periplasm protecting them from aggregation. The chaperones then deliver OMPs to a highly conserved outer membrane assembly site termed the Bam complex. In E. coli, the Bam complex is composed of an essential OMP, BamA, and four associated OM lipoproteins, BamBCDE, one of which, BamD, is also essential. Here we provide an overview of what we know about the process of OMP assembly and outline the various hypotheses that have been proposed to explain how proteins might be integrated into the asymmetric OM lipid bilayer in an environment that lacks obvious energy sources. In addition, we describe the envelope stress responses that ensure the fidelity of OM biogenesis and how factors, such as phage and certain toxins, have coopted this essential machine to gain entry into the cell.

The type II secretion system (T2SS) is used by Escherichia coli and other gram-negative bacteria to translocate many proteins, including toxins and proteases, across the outer membrane of the cell and into the extracellular space. Depending on the bacterial species, between 12 and 15 genes have been identified that make up a T2SS operon. T2SSs are widespread among gram-negative bacteria, and most E. coli appear to possess one or two complete T2SS operons. Once expressed, the multiple protein components that form the T2S system are localized in both the inner and outer membranes, where they assemble into an apparatus that spans the cell envelope. This apparatus supports the secretion of numerous virulence factors; and therefore secretion via this pathway is regarded in many organisms as a major virulence mechanism. Here, we review several of the known E. coli T2S substrates that have proven to be critical for the survival and pathogenicity of these bacteria. Recent structural and biochemical information is also reviewed that has improved our current understanding of how the T2S apparatus functions; also reviewed is the role that individual proteins play in this complex system.

The autotransporter and two-partner secretion (TPS) pathways are used by E. coli and many other Gram-negative bacteria to delivervirulence factors into the extracellular milieu.Autotransporters arecomprised of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that anchors the protein to the outer membrane and facilitates passenger domain secretion. In the TPS pathway, a secreted polypeptide ("exoprotein") is coordinately expressed with an outer membrane protein that serves as a dedicated transporter. Bothpathways are often grouped together under the heading "type V secretion" because they have many features in common and are used for the secretion of structurally related polypeptides, but it is likely that theyhave distinct evolutionary origins. Although it was proposed many years ago that autotransporterpassenger domains are transported across the outer membrane through a channel formed by the covalently linked β domain, there is increasing evidence that additional factors are involved in the translocation reaction. Furthermore, details of the mechanism of protein secretion through the TPS pathway are only beginning to emerge. In this chapter I discussour current understanding ofboth early and late steps in the biogenesis of polypeptides secreted through type V pathways and current modelsofthe mechanism of secretion.

The type VI secretion system (T6SS) is a multiprotein complex widespread in Proteobacteria and dedicated to the delivery of toxins into both prokaryotic and eukaryotic cells. It thus participates in interbacterial competition as well as pathogenesis. The T6SS is a contractile weapon, related to the injection apparatus of contractile tailed bacteriophages. Basically, it assembles an inner tube wrapped by a sheath-like structure and anchored to the cell envelope via a membrane complex. The energy released by the contraction of the sheath propels the inner tube through the membrane channel and toward the target cell. Although the assembly and the mechanism of action are conserved across species, the repertoire of secreted toxins and the diversity of the regulatory mechanisms and of target cells make the T6SS a highly versatile secretion system. The T6SS is particularly represented in Escherichia coli pathotypes and Salmonella serotypes. In this review we summarize the current knowledge regarding the prevalence, the assembly, the regulation, and the roles of the T6SS in E. coli, Salmonella, and related species.

In Escherichia coli, proteins found in the periplasm or the outer membrane are exported from the cytoplasm by the general secretory, Sec, system before they acquire stably folded structure. This dynamic process involves intricate interactions among cytoplasmic and membrane proteins, both peripheral and integral, as well as lipids. In vivo, both ATP hydrolysis and proton motive force are required. Here, we review the Sec system from the inception of the field through early 2016, including biochemical, genetic, and structural data.

Escherichia coli and other Gram-negative and -positive bacteria employ type IV secretion systems (T4SSs) to translocate DNA and protein substrates, generally by contact-dependent mechanisms, to other cells. The T4SSs functionally encompass two major subfamilies, the conjugation systems and the effector translocators. The conjugation systems are responsible for interbacterial transfer of antibiotic resistance genes, virulence determinants, and genes encoding other traits of potential benefit to the bacterial host. The effector translocators are used by many Gram-negative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection. In E. coli and other species of Enterobacteriaceae, T4SSs identified to date function exclusively in conjugative DNA transfer. In these species, the plasmid-encoded systems can be classified as the P, F, and I types. The P-type systems are the simplest in terms of subunit composition and architecture, and members of this subfamily share features in common with the paradigmatic Agrobacterium tumefaciens VirB/VirD4 T4SS. This review will summarize our current knowledge of the E. coli systems and the A. tumefaciens P-type system, with emphasis on the structural diversity of the T4SSs. Ancestral P-, F-, and I-type systems were adapted throughout evolution to yield the extant effector translocators, and information about well-characterized effector translocators also is included to further illustrate the adaptive and mosaic nature of these highly versatile machines.

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process.

Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope “tunnel” is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

The insertion and assembly of proteins into the inner membrane of bacteria are crucial for many cellular processes, including cellular respiration, signal transduction, and ion and pH homeostasis. This process requires efficient membrane targeting and insertion of proteins into the lipid bilayer in their correct orientation and proper conformation. Playing center stage in these events are the targeting components, signal recognition particle (SRP) and the SRP receptor FtsY, as well as the insertion components, the Sec translocon and the YidC insertase. Here, we will discuss new insights provided from the recent high-resolution structures of these proteins. In addition, we will review the mechanism by which a variety of proteins with different topologies are inserted into the inner membrane of Gram-negative bacteria. Finally, we report on the energetics of this process and provide information on how membrane insertion occurs in Gram-positive bacteria and Archaea. It should be noted that most of what we know about membrane protein assembly in bacteria is based on studies conducted in Escherichia coli.

DNA

In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC, the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli, in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC, were identified. The DnaA protein is the bacterial initiator, and in E. coli, the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC, the ordered assembly and disassembly of a multi-DnaA complex at oriC, and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.

This review describes the components of the Escherichia coli replisome and the dynamic process in which they function and interact under normal conditions. It also briefly describes the behavior of the replisome during situations in which normal replication fork movement is disturbed, such as when the replication fork collides with sites of DNA damage. E. coli DNA Pol III was isolated first from a polA mutant E. coli strain that lacked the relatively abundant DNA Pol I activity. Further biochemical studies, and the use of double mutant strains, revealed Pol III to be the replicative DNA polymerase essential to cell viability. In a replisome, DnaG primase must interact with DnaB for activity, and this constraint ensures that new RNA primers localize to the replication fork. The leading strand polymerase continually synthesizes DNA in the direction of the replication fork, whereas the lagging-strand polymerase synthesizes short, discontinuous Okazaki fragments in the opposite direction. Discontinuous lagging-strand synthesis requires that the polymerase rapidly dissociate from each new completed Okazaki fragment in order to begin the extension of a new RNA primer. Lesion bypass can be thought of as a two-step reaction that starts with the incorporation of a nucleotide opposite the lesion, followed by the extension of the resulting distorted primer terminus. A remarkable property of E. coli, and many other eubacterial organisms, is the speed at which it propagates. Rapid cell division requires the presence of an extremely efficient replication machinery for the rapid and faithful duplication of the genome.

The DNA of Escherichia coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases, and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during the repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and the regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation, although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential, and, in C. crescentus, it is important for temporal gene expression, which, in turn, is required for coordinating chromosome initiation, replication, and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage, decrease transformation frequency in certain bacteria, and decrease the stability of short direct repeats and are necessary for site-directed mutagenesis and to probe eukaryotic structure and function.

DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry, and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB), and III (dnaQ/mutD); Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG), and X (exoX); the RecBCD, RecJ, and RecE exonucleases; SbcCD endo/exonucleases; the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo); and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.

DNA and RNA helicases are organized into six superfamilies of enzymes on the basis of sequence alignments, biochemical data, and available crystal structures. DNA helicases, members of which are found in each of the superfamilies, are an essential group of motor proteins that unwind DNA duplexes into their component single strands in a process that is coupled to the hydrolysis of nucleoside 5'-triphosphates. The purpose of this DNA unwinding is to provide nascent, single-stranded DNA (ssDNA) for the processes of DNA repair, replication, and recombination. Not surprisingly, DNA helicases share common biochemical properties that include the binding of single- and double-stranded DNA, nucleoside 5'-triphosphate binding and hydrolysis, and nucleoside 5'-triphosphate hydrolysis-coupled, polar unwinding of duplex DNA. These enzymes participate in every aspect of DNA metabolism due to the requirement for transient separation of small regions of the duplex genome into its component strands so that replication, recombination, and repair can occur. In Escherichia coli, there are currently twelve DNA helicases that perform a variety of tasks ranging from simple strand separation at the replication fork to more sophisticated processes in DNA repair and genetic recombination. In this chapter, the superfamily classification, role(s) in DNA metabolism, effects of mutations, biochemical analysis, oligomeric nature, and interacting partner proteins of each of the twelve DNA helicases are discussed.

DNA topoisomerases are enzymes that control the topology of DNA in all cells. There are two types, I and II, classified according to whether they make transient single- or double-stranded breaks in DNA. Their reactions generally involve the passage of a single- or double-strand segment of DNA through this transient break, stabilized by DNA-protein covalent bonds. All topoisomerases can relax DNA, but DNA gyrase, present in all bacteria, can also introduce supercoils into DNA. Because of their essentiality in all cells and the fact that their reactions proceed via DNA breaks, topoisomerases have become important drug targets; the bacterial enzymes are key targets for antibacterial agents. This article discusses the structure and mechanism of topoisomerases and their roles in the bacterial cell. Targeting of the bacterial topoisomerases by inhibitors, including antibiotics in clinical use, is also discussed.

All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase. In these situations, cells are forced to choose between recombination-mediated "damage avoidance" pathways or a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions but also bases being (mis)incorporated downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV, and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases.

Early research on the origins and mechanisms of mutation led to the establishment of the dogma that, in the absence of external forces, spontaneous mutation rates are constant. However, recent results from a variety of experimental systems suggest that mutation rates can increase in response to selective pressures. This chapter summarizes data demonstrating that,under stressful conditions, Escherichia coli and Salmonella can increase the likelihood of beneficial mutations by modulating their potential for genetic change.Several experimental systems used to study stress-induced mutagenesis are discussed, with special emphasison the Foster-Cairns system for "adaptive mutation" in E. coli and Salmonella. Examples from other model systems are given to illustrate that stress-induced mutagenesis is a natural and general phenomenon that is not confined to enteric bacteria. Finally, some of the controversy in the field of stress-induced mutagenesis is summarized and discussed, and a perspective on the current state of the field is provided.

Cellular DNA is constantly challenged by various endogenous and exogenous genotoxic factors that inevitably lead to DNA damage: structural and chemical modifications of primary DNA sequence. These DNA lesions are either cytotoxic, because they block DNA replication and transcription, or mutagenic due to the miscoding nature of the DNA modifications, or both, and are believed to contribute to cell lethality and mutagenesis. Studies on DNA repair in Escherichia coli spearheaded formulation of principal strategies to counteract DNA damage and mutagenesis, such as: direct lesion reversal, DNA excision repair, mismatch and recombinational repair and genotoxic stress signalling pathways. These DNA repair pathways are universal among cellular organisms. Mechanistic principles used for each repair strategies are fundamentally different. Direct lesion reversal removes DNA damage without need for excision and de novo DNA synthesis, whereas DNA excision repair that includes pathways such as base excision, nucleotide excision, alternative excision and mismatch repair, proceeds through phosphodiester bond breakage, de novo DNA synthesis and ligation. Cell signalling systems, such as adaptive and oxidative stress responses, although not DNA repair pathways per se, are nevertheless essential to counteract DNA damage and mutagenesis. The present review focuses on the nature of DNA damage, direct lesion reversal, DNA excision repair pathways and adaptive and oxidative stress responses in E. coli.

DNA mismatch repair (MMR) corrects replication errors in newly synthesized DNA. It also has an antirecombination action on heteroduplexes that contain similar but not identical sequences. This review focuses on the genetics and development of MMR and not on the latest biochemical mechanisms. The main focus is on MMR in Escherichia coli, but examples from Streptococcuspneumoniae and Bacillussubtilis have also been included. In most organisms, only MutS (detects mismatches) and MutL (an endonuclease) and a single exonucleaseare present. How this system discriminates between newlysynthesized and parental DNA strands is not clear. In E. coli and its relatives, however, Dam methylation is an integral part of MMR and is the basis for strand discrimination. A dedicated site-specific endonuclease, MutH, is present, andMutL has no endonuclease activity; four exonucleases can participate in MMR. Although it might seem that the accumulated wealth of genetic and biochemical data has given us a detailed picture of the mechanism of MMR in E. coli, the existence of three competing models to explain the initiation phase indicates the complexity of the system. The mechanism of the antirecombination action of MMR is largely unknown, but only MutS and MutL appear to be necessary. A primary site of action appears to be on RecA, although subsequent steps of the recombination process can also be inhibited. In this review, the genetics of Very Short Patch (VSP) repair of T/G mismatches arising from deamination of 5-methylcytosineresidues is also discussed.

Homologous recombination is the most complex of all recombination events that shape genomes and produce material for evolution. Homologous recombination events are exchanges between DNA molecules in the lengthy regions of shared identity, catalyzed by a group of dedicated enzymes. There is a variety of experimental systems in Escherichia coli and Salmonella to detect homologous recombination events of several different kinds. Genetic analysis of homologous recombination reveals three separate phases of this process: pre-synapsis (the early phase), synapsis (homologous strand exchange), and post-synapsis (the late phase). In E. coli, there are at least two independent pathway of the early phase and at least two independent pathways of the late phase. All this complexity is incongruent with the originally ascribed role of homologous recombination as accelerator of genome evolution: there is simply not enough duplication and repetition in enterobacterial genomes for homologous recombination to have a detectable evolutionary role and therefore not enough selection to maintain such a complexity. At the same time, the mechanisms of homologous recombination are uniquely suited for repair of complex DNA lesions called chromosomal lesions. In fact, the two major classes of chromosomal lesions are recognized and processed by the two individual pathways at the early phase of homologous recombination. It follows, therefore, that homologous recombination events are occasional reflections of the continual recombinational repair, made possible in cases of natural or artificial genome redundancy.

Homologous recombination is an ubiquitous process that shapes genomes and repairs DNA damage. The reaction is classically divided into three phases: presynaptic, synaptic, and postsynaptic. In Escherichia coli, the presynaptic phase involves either RecBCD or RecFOR proteins, which act on DNA double-stranded ends and DNA single-stranded gaps, respectively; the central synaptic steps are catalyzed by the ubiquitous DNA-binding protein RecA; and the postsynaptic phase involves either RuvABC or RecG proteins, which catalyze branch-migration and, in the case of RuvABC, the cleavage of Holliday junctions. Here, we review the biochemical properties of these molecular machines and analyze how, in light of these properties, the phenotypes of null mutants allow us to define their biological function(s). The consequences of point mutations on the biochemical properties of recombination enzymes and on cell phenotypes help refine the molecular mechanisms of action and the biological roles of recombination proteins. Given the high level of conservation of key proteins like RecA and the conservation of the principles of action of all recombination proteins, the deep knowledge acquired during decades of studies of homologous recombination in bacteria is the foundation of our present understanding of the processes that govern genome stability and evolution in all living organisms.

RNA

Promoter escape is the process that an initiated RNA polymerase (RNAP) molecule undergoes to achieve the initiation-elongation transition. Having made this transition, an RNAP molecule would be relinquished from its promoter hold to perform productive (full-length) transcription. Prior to the transition, this process is accompanied by abortive RNA formation—the amount and pattern of which is controlled by the promoter sequence information. Qualitative and quantitative analysis of abortive/productive transcription from several Escherichia coli promoters and their sequence variants led to the understanding that a strong (RNAP-binding) promoter is more likely to be rate limited (during transcription initiation) at the escape step and produce abortive transcripts. Of the two subelements in a promoter, the PRR (the core Promoter Recognition Region) was found to set the initiation frequency and the rate-limiting step, while the ITS (the Initial Transcribed Sequence region) modulated the ratio of abortive versus productive transcription. The highly abortive behavior of E. coli RNAP could be ameliorated by the presence of Gre (transcript cleavage stimulatory) factor(s), linking the first step in abortive RNA formation by the initial transcribing complexes (ITC) to RNAP backtracking. The discovery that translocation during the initiation stage occurs via DNA scrunching provided the source of energy that converts each ITC into a highly unstable "stressed intermediate." Mapping all of the biochemical information onto an X-ray crystallographic structural model of an open complex gave rise to a plausible mechanism of transcription initiation. The chapter concludes with contemplations of the kinetics and thermodynamics of abortive initiation-promoter escape.

The highly conserved Nus factors of bacteria were discovered as essential host proteins for the growth of temperate phage λ in Escherichia coli. Later, their essentiality and functions in transcription, translation, and, more recently, in DNA repair have been elucidated. Close involvement of these factors in various gene networks and circuits is also emerging from recent genomic studies. We have described a detailed overview of their biochemistry, structures, and various cellular functions, as well as their interactions with other macromolecules. Towards the end, we have envisaged different uncharted areas of studies with these factors, including their participation in pathogenicity.

This review provides a description of the known Escherichia coli ribonucleases (RNases), focusing on their structures, catalytic properties, genes, physiological roles, and possible regulation. Currently, eight E. coli exoribonucleases are known. These are RNases II, R, D, T, PH, BN, polynucleotide phosphorylase (PNPase), and oligoribonuclease (ORNase). Based on sequence analysis and catalytic properties, the eight exoribonucleases have been grouped into four families. These are the RNR family, including RNase II and RNase R; the DEDD family, including RNase D, RNase T, and ORNase; the RBN family, consisting of RNase BN; and the PDX family, including PNPase and RNase PH. Seven well-characterized endoribonucleases are known in E. coli. These are RNases I, III, P, E, G, HI, and HII. Homologues to most of these enzymes are also present in Salmonella. Most of the endoribonucleases cleave RNA in the presence of divalent cations, producing fragments with 3'-hydroxyl and 5'-phosphate termini. RNase H selectively hydrolyzes the RNA strand of RNA?DNA hybrids. Members of the RNase H family are widely distributed among prokaryotic and eukaryotic organisms in three distinct lineages, RNases HI, HII, and HIII. It is likely that E. coli contains additional endoribonucleases that have not yet been characterized. First of all, endonucleolytic activities are needed for certain known processes that cannot be attributed to any of the known enzymes. Second, homologues of known endoribonucleases are present in E. coli. Third, endonucleolytic activities have been observed in cell extracts that have different properties from known enzymes.

This chapter discusses several topics relating to the mechanisms of mRNA decay. These topics include the following: important physical properties of mRNA molecules that can alter their stability; methods for determining mRNA half-lives; the genetics and biochemistry of proteins and enzymes involved in mRNA decay; posttranscriptional modification of mRNAs; the cellular location of the mRNA decay apparatus; regulation of mRNA decay; the relationships among mRNA decay, tRNA maturation, and ribosomal RNA processing; and biochemical models for mRNA decay. Escherichia coli has multiple pathways for ensuring the effective decay of mRNAs and mRNA decay is closely linked to the cell's overall RNA metabolism. Finally, the chapter highlights important unanswered questions regarding both the mechanism and importance of mRNA decay.

Complex Lipids and Carbohydrates

Peptidoglycan (PG) recycling allows Escherichia coli to reuse the massive amounts of sacculus components that are released during elongation. Goodell and Schwarz, in 1985, labeled E. coli cells with 3H-diaminopimelic acid (DAP) and chased. During the chase, the DAP pool dropped dramatically, whereas the precursor pool dropped only slightly. This could only occur if DAP from the sacculi was being used to produce more precursor. They calculated that the cells were recycling about 45% of their wall DAP (actually, 60% of the side walls, since the poles are stable). Thus, recycling was discovered. Goodell went on to show that the tripeptide, L-Ala-D-Glu-DAP, could be taken up via opp and used directly to form PG. It was subsequently shown that uptake was predominantly via a permease, AmpG, that was specific for GlcNAc-anhMurNAc with attached peptides. Eleven genes have been identified which appear to have as their sole function the recovery of degradation products from PG. PG represents only 2.5% of the cell mass, so the reason for this investment in recycling is obscure. Recycling enzymes exist that are specific for every bond in the principal product taken up by AmpG, namely, GlcNAc-anh-MurNAc-tetrapeptide. However, most of the tripeptide, L-Ala-D-Glu-DAP, is used by murein peptide ligase (Mpl) to form the precursor intermediate UDP-MurNAc-tripeptide. anh-MurNAc can be converted to GlcNAc by a two-step process and thus is available for use. Surprisingly, in the absence of AmpD, an enzyme that cleaves the anh-MurNAc-L-Ala bond, anh-MurNAc-tripeptide accumulates, resulting in induction of beta-lactamase. However, this has nothing to do with the induction of beta-lactamase by beta-lactam antibiotics. Uehara, Suefuji, and Park (unpublished data) have some evidence suggesting that murein pentapeptide may be involved. The presence of orthologs suggests that recycling also exists in many Gram-negative bacteria. Surprisingly, the ortholog search also revealed that all mammals may have an AmpG ortholog! Hence, mammalian AmpG may be involved in the process of innate immunity.

Undecaprenyl phosphate (C55-P) is an essential 55-carbon long-chain isoprene lipidinvolved in the biogenesis of bacterial cell wall carbohydrate polymers: peptidoglycan, O antigen, teichoic acids, and other cell surface polymers. It functions as a lipid carrier that allows the traffic of sugar intermediates across the plasma membrane, towards the periplasm,where the polymerization of the different cellwall components occurs. At the end of these processes, the lipid is released in a pyrophosphate form (C55-PP). C55-P arises from the dephosphorylation of C55-PP, which itself originates from either a recycling event or a de novo synthesis. In Escherichia coli, the formation of C55-PP is catalyzed by the essential UppS synthase, a soluble cis-prenyltransferase, whichadds eight isoprene units ontofarnesyl pyrophosphate. Severalapo- and halo-UppSthree-dimensional structures have provided a high level of understanding of this enzymatic step. The following dephosphorylationstep is required before the lipid carrier can accept a sugar unit at the cytoplasmic face of the membrane. Four integralmembrane proteins have been shown to catalyzethis reaction in E. coli:BacA and three members of the PAP2 super-family:YbjG, LpxT, and PgpB. None of these enzymes is essential,but the simultaneous inactivation of bacA, ybjG, and pgpB genes gave rise to a lethal phenotype, raising the question of the relevance of such a redundancy of activity. It was alsorecently shown that LpxTcatalyzes the specific transfer of the phosphate group arising from C55-PP to the lipidA moiety of lipopolysaccharides, leading to a lipid-A 1-diphosphate form whichaccounts for one-third of the total lipidA in wild-type E. coli cells. The active sites of LpxT, PgpB,andYbjG were shown to face the periplasm, suggesting that PAP2 enzymes arerather involved in C55-PP recycling. These recent discoveries have opened the way to the elucidation of the functional and structural characterization of these different phosphatases.

This review focuses on the cold shock response of Escherichia coli. Change in temperature is one of the most common stresses that an organism encounters in nature. Temperature downshift affects the cell on various levels: (i) decrease in the membrane fluidity; (ii) stabilization of the secondary structures of RNA and DNA; (iii) slow or inefficient protein folding; (iv) reduced ribosome function, affecting translation of non-cold shock proteins; (v) increased negative supercoiling of DNA; and (vi) accumulation of various sugars. Cold shock proteins and certain sugars play a key role in dealing with the initial detrimental effect of cold shock and maintaining the continued growth of the organism at low temperature. CspA is the major cold shock protein of E. coli, and its homologues are found to be widespread among bacteria, including psychrophilic, psychrotrophic, mesophilic, and thermophilic bacteria, but are not found in archaea or cyanobacteria. Significant, albeit transient, stabilization of the cspA mRNA immediately following temperature downshift is mainly responsible for its cold shock induction. Various approaches were used in studies to detect cold shock induction of cspA mRNA. Sugars are shown to confer protection to cells undergoing cold shock. The study of the cold shock response has implications in basic and health-related research as well as in commercial applications. The cold shock response is elicited by all types of bacteria and affects these bacteria at various levels, such as cell membrane, transcription, translation, and metabolism.

All organisms possess a diverse set of genetic programs that are used to alter cellular physiology in response to environmental cues. The gram-negative bacterium Escherichia coli induces a gene regulatory network known as the “SOS response” following exposure to DNA damage, replication fork arrest, and a myriad of other environmental stresses. For over 50 years, E. coli has served as the paradigm for our understanding of the transcriptional and physiological changes that occur after DNA damage. In this chapter, we summarize the current view of the SOS response and discuss how this genetic circuit is regulated. In addition to examining the E. coli SOS response, we include a discussion of the SOS regulatory networks found in other bacteria to provide a broad perspective on the mechanism and diverse physiological responses that ensueto protect cells and maintain genome integrity.

The ancestors of Escherichia coli and Salmonella ultimately evolved to thrive in air-saturated liquids, in which oxygen levels reach 210 μM at 37°C. However, in 1976 Brown and colleagues reported that some sensitivity persists: growth defects still become apparent when hyperoxia is imposed on cultures of E. coli. This residual vulnerability was important in that it raised the prospect that normal levels of oxygen might also injure bacteria, albeit at reduced rates that are not overtly toxic. The intent of this article is both to describe the threat that molecular oxygen poses for bacteria and to detail what we currently understand about the strategies by which E. coli and Salmonella defend themselves against it. E. coli mutants that lack either superoxide dismutases or catalases and peroxidases exhibit a variety of growth defects. These phenotypes constitute the best evidence that aerobic cells continually generate intracellular superoxide and hydrogen peroxide at potentially lethal doses. Superoxide has reduction potentials that allow it to serve in vitro as either a weak univalent reductant or a stronger univalent oxidant. The addition of micromolar hydrogen peroxide to lab media will immediately block the growth of most cells, and protracted exposure will result in the loss of viability. The need for inducible antioxidant systems seems especially obvious for enteric bacteria, which move quickly from the anaerobic gut to fully aerobic surface waters or even to ROS-perfused phagolysosomes. E. coli and Salmonella have provided two paradigmatic models of oxidative-stress responses: the SoxRS and OxyR systems.

Escherichia coli and Salmonella encounter osmotic pressure variations in natural environments that include host tissues, food, soil, and water. Osmotic stress causes water to flow into or out of cells, changing their structure, physics, and chemistry in ways that perturb cell functions. E. coli and Salmonella limit osmotically induced water fluxes by accumulating and releasing electrolytes and small organic solutes, some denoted compatible solutes because they accumulate to high levels without disturbing cell functions. Osmotic upshifts inhibit membrane-based energy transduction and macromolecule synthesis while activating existing osmoregulatory systems and specifically inducing osmoregulatory genes. The osmoregulatory response depends on the availability of osmoprotectants (exogenous organic compounds that can be taken up to become compatible solutes). Without osmoprotectants, K+ accumulates with counterion glutamate, and compatible solute trehalose is synthesized. Available osmoprotectants are taken up via transporters ProP, ProU, BetT, and BetU. The resulting compatible solute accumulation attenuates the K+ glutamate response and more effectively restores cell hydration and growth. Osmotic downshifts abruptly increase turgor pressure and strain the cytoplasmic membrane. Mechanosensitive channels like MscS and MscL open to allow nonspecific solute efflux and forestall cell lysis. Research frontiers include (i) the osmoadaptive remodeling of cell structure, (ii) the mechanisms by which osmotic stress alters gene expression, (iii) the mechanisms by which transporters and channels detect and respond to osmotic pressure changes, (iv) the coordination of osmoregulatory programs and selection of available osmoprotectants, and (v) the roles played by osmoregulatory mechanisms as E. coli and Salmonella survive or thrive in their natural environments.

The gram-negative bacterial envelope is a complex extracytoplasmic compartment responsible for numerous cellular processes. Among its most important functions is its service as the protective layer separating the cytoplasmic space from the ever-changing external environment. To adapt to the diverse conditions encountered both in the environment and within the mammalian host, Escherichia coli and Salmonella species have evolved six independent envelope stress response systems . This review reviews the sE response, the CpxAR and BaeSR two-component systems (TCS) , the phage shock protein response, and the Rcs phosphorelay system. These five signal transduction pathways represent the most studied of the six known stress responses. The signal for adhesion to abiotic surfaces enters the pathway through the novel outer membrane lipoprotein NlpE, and activation on entry into the exponential phase of growth occurs independently of CpxA . Adhesion could disrupt NlpE causing unfolding of its unstable N-terminal domain, leading to activation of the Cpx response. The most recent class of genes added to the Cpx regulon includes those involved in copper homeostasis. Two separate microarray experiments revealed that exposure of E. coli cells to high levels of external copper leads to upregulation of several Cpx regulon members. The BaeSR TCS has also been shown to mediate drug resistance in Salmonella. Similar to E. coli, the Bae pathway of Salmonella enterica mediates resistance to oxacillin, novobiocin, deoxycholate, β-lactams, and indole.

In their stressful natural environments, bacteria often are in stationary phase and use their limited resources for maintenance and stress survival. Underlying this activity is the general stress response, which in Escherichia coli depends on the σS (RpoS) subunit of RNA polymerase. σS is closely related to the vegetative sigma factor σ70 (RpoD), and these two sigmas recognize similar but not identical promoter sequences. During the postexponential phase and entry into stationary phase, σS is induced by a fine-tuned combination of transcriptional, translational, and proteolytic control. In addition, regulatory "short-cuts" to high cellular σS levels, which mainly rely on the rapid inhibition of σS proteolysis, are triggered by sudden starvation for various nutrients and other stressful shift conditons. σS directly or indirectly activates more than 500 genes. Additional signal input is integrated by σS cooperating with various transcription factors in complex cascades and feedforward loops. Target gene products have stress-protective functions, redirect metabolism, affect cell envelope and cell shape, are involved in biofilm formation or pathogenesis, or can increased stationary phase and stress-induced mutagenesis. This review summarizes these diverse functions and the amazingly complex regulation of σS. At the molecular level, these processes are integrated with the partitioning of global transcription space by sigma factor competition for RNA polymerase core enzyme and signaling by nucleotide second messengers that include cAMP, (p)ppGpp, and c-di-GMP. Physiologically, σS is the key player in choosing between a lifestyle associated with postexponential growth based on nutrient scavenging and motility and a lifestyle focused on maintenance, strong stress resistance, and increased adhesiveness. Finally, research with other proteobacteria is beginning to reveal how evolution has further adapted function and regulation of σS to specific environmental niches.

Among all the systems developed by enterobacteria to face osmotic stress, only osmoregulated periplasmic glucans (OPGs) were found to be modulated during osmotic fluxes. First detected in 1973 by E.P. Kennedy’s group in a study of phospholipid turnover in Escherichia coli, OPGs have been shown across alpha, beta, and gamma subdivisions of the proteobacteria. Discovery of OPG-like compounds in the epsilon subdivision strongly suggested that the presence of periplasmic glucans is essential for almost all proteobacteria. This article offers an overview of the different classes of OPGs. Then, the biosynthesis of OPGs and their regulation in E. coli and other species are discussed. Finally, the biological role of OPGs is developed. Beyond structural function, OPGs are involved in pathogenicity, in particular, by playing a role in signal transduction pathways. Recently, OPG synthesis proteins have been suggested to control cell division and growth rate.

Domain 6

Citation: David M. Gordon. The influence of ecological factors on the distribution and the genetic structure of escherichia coli. doi:10.1128/ecosalplus.6.4.1

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This review focuses on recent data concerning the ecological factors that determine the distribution of Escherichia coli and the genetic structures of naturally occurring E. coli populations. It summarizes some of the older literature concerning the dynamics of E. coli populations within a host and poses some questions that arise from our more recently acquired understanding of the factors affecting the genetic structures of E. coli populations. Multilocus enzyme electrophoresis (MLEE) studies indicate that E. coli, relative to other members of the family Enterobacteriaceae, exhibits a moderate degree of genetic diversity. The existence of subspecific structure in E. coli has for the most part been determined by largely neutral in its effects on the fitness of a strain. The consequences for E. coli of the transition between its primary and secondary habitats are of considerable practical significance for water quality assessment and disease transmission. E. coli causes a significant fraction of human bacterial disease and is responsible for two main types of disease in humans and domestic animals: diarrheal disease and extraintestinal infections. The observed distribution of strains from the different E. coli genetic groups indicates that they have different life history tactics and ecological niches. A and B1 strains appear to be generalists, as they can be recovered from any vertebrate group. Group B2 and D strains appear to be more specialized, as they are largely restricted to endothermic vertebrates.

Over the past 120 to 160 million years, the genus Salmonella has evolved into a complex group of more than 2,300 genetically and phenotypically diverse serovars. Members of this genus are able to infect a wide diversity of vertebrate and invertebrate hosts; disease manifestations in humans range from gastroenteritis to typhoid fever. The evolution of the genus Salmonella and the divergence and radiation of particular lineages within this group have resulted from selection acting on new genetic variation generated by events such as the gain, loss, and/or rearrangement of genetic material. These types of genetic events have contributed to the speciation of Salmonella from its ancestral association with cold-blood animals to a pathogen of warm-blooded hosts. Moreover, adaptive radiation due to changes in gene content within S. enterica subspecies I has impacted host specificity and aided in the selection of host-restricted, host-adapted, and non-host-adapted serovars. In addition to the genetic diversity important for the wide phenotypic heterogeneity within the genus, a subset of core Salmonella-specific genes present in all Salmonella species and serovars has been identified that may contribute to the conserved aspects of the lifestyle of this microorganism, including the ability to survive in nutrient-poor nonhost environments such as soil and water. Whole-genome comparisons of isolates differing in host range and virulence will continue to elucidate the genetic mechanisms that have contributed to the evolution and diverse ecology of the genus Salmonella.

Microbes produce an extraordinary array of microbial defense systems. These include broad-spectrum classical antibiotics critical to human health concerns; metabolic by-products, such as the lactic acids produced by lactobacilli; lytic agents, such as lysozymes found in many foods; and numerous types of protein exotoxins and bacteriocins. The abundance and diversity of this biological arsenal are clear. Lactic acid production is a defining trait of lactic acid bacteria. Bacteriocins are found in almost every bacterial species examined to date, and within a species, tens or even hundreds of different kinds of bacteriocins are produced. Halobacteria universally produce their own version of bacteriocins, the halocins. Streptomycetes commonly produce broad-spectrum antibiotics. It is clear that microbes invest considerable energy in the production and elaboration of antimicrobial mechanisms. What is less clear is how such diversity arose and what roles these biological weapons play in microbial communities. One family of microbial defense systems, the bacteriocins, has served as a model for exploring evolutionary and ecological questions. In this review, current knowledge of how the extraordinary range of bacteriocin diversity arose and is maintained in one species of bacteria, Escherichia coli, is assessed and the role these toxins play in mediating microbial dynamics is discussed.

The classical experiments of Luria and Delbrück showed convincingly that mutations exist before selection and do not contribute to the creation of mutations when selection is lethal. In contrast, when nonlethal selections are used,measuring mutation rates and separating the effects of mutation and selection are difficult and require methods to fully exclude growth after selection has been applied. Although many claims of stress-induced mutagenesis have been made, it is difficult to exclude the influence of growth under nonlethal selection conditions in accounting for the observed increases in mutant frequency. Instead, for many of the studied experimental systems the increase in mutant frequency can be explainedbetter by the ability of selection to detect small differences in growth rate caused by common small effect mutations. A verycommon mutant class,found in response to many different types of selective regimensin which increased gene dosage can resolve the problem, is gene amplification. In the well-studiedlac system of Cairns and Foster, the apparent increase in Lac+revertants can be explained by high-level amplification of the lac operon and the increased probability for a reversion mutation to occur in any one of the amplified copies. The associated increase in general mutation rate observed in revertant cells in that system is an artifact caused by the coincidental co-amplification of the nearby dinB gene (encoding the error-prone DNA polymerase IV) on the particular plasmid used for these experiments. Apart from the lac system, similar gene amplification processes have been described for adaptation to toxic drugs, growth in host cells, and various nutrient limitations.

In 2009, five monophyletic Escherichia clades were described and referred to as “cryptic” based on the inability to distinguish them from representative E. coli isolates using diagnostic biochemical reactions. Since this original publication, a number of studies have explored the genomic, transcriptomic, and phenotypic diversity of cryptic clade isolates to better understand their phylogenetic, physiological, and ecological distinctiveness with respect to previously named Escherichia species. This chapter reviews the original discovery of the cryptic clades, discusses available evidence that some are environmentally adapted, and evaluates current support for taxonomic designations of these microorganisms. The importance of these clades to clinical research, epidemiology, population genetics, and microbial speciation is also discussed.

Domain 7

Genetics and Genetic Tools

Editor: James M. Slauch

Bacteriophage

The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

This review focuses on the membrane-associated structures present at cell-cell contact sites during bacterial conjugation. These transfer proteins/structures have roles in the formation and stabilization of mating contacts and ultimately the passage of substrate across the cell envelope between two bacterial cells. The review presents evidence for the dynamic interaction between donor and recipient cells, including the assembly of a transmembrane protein complex, and concludes with a refined model for the mechanism of bacterial conjugation. Bacterial conjugation, in addition to being a mechanism for genome evolution, can be considered as a mechanism for macromolecular secretion. In particular, plasmid-conjugative transfer is classified as a type IV secretion (T4S) system and represents the only known bacterial system for secretion of DNA. In all known conjugative transfer systems, a multitude of proteins are required for both plasmid transfer and pilus production. The plasmids discussed in the review include the F factor; the P group of plasmids, including RP4 and R751 (rigid); and the H plasmid group, including R27 (also thick flexible). With the LacI-GFP/lacO system, the F, P, and H plasmids were observed to reside at well-defined positions located at the mid and quarter-cell positions of Escherichia coli throughout the vegetative cycle. In this review, recent observations based on bacterial cell biology techniques, including visualization of plasmid DNA and proteins at the subcellular level, have been combined with electron and light microscopy studies of mating cells to create an integrated overview of gram-negative bacterial conjugation, a concept referred to as the conjugative cycle.

Escherichia coli K-12 and Salmonella enterica serovar Typhimurium LT2 became standard organisms for genetic analysis during the Truman administration. Half a century later, genetic analysis with these strains had become an art form, interpreted through 23 articles in the ambitious two-volume masterpiece edited by the late Fred Neidhardt and colleagues. These legacy articles now are available through EcoSal Plus, so as to inform and inspire contemporary genetic analyses in these standard organisms and their relatives.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors: adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17, and F18 fimbriae. Once established in the animal small intestine, ETEC produce enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes: heat-labile toxins that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This review describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics, and the identification of potential new targets by genomics are presented in the context of animal ETEC.

Epidemiological studies have implicated enteroaggregative Escherichia coli (EAEC) strains in acute and persistent diarrhea in children, in food-borne diarrhea outbreaks, and in traveler's diarrhea, and this group is recognized as an emerging pathotype of enteric disease. Diffusely adherent E. coli (DAEC) have been implicated as a cause of diarrhea, especially in children more than 2 years old, in both developing and developed countries. Although EAEC and DAEC strains appear to have different molecular equipment for attachment to host cell surfaces, identification and characterization of the gene clusters encoding adherence evidenced close relatedness between those determinants most frequently detected in isolates belonging to these two pathotypes of diarrheagenic E. coli. DAEC strains are a heterogeneous group of E. coli isolates, many of which express the related so-called Dr adhesins. The single designation is based on the identification of one similar cellular receptor for all these proteins. Although structurally different, they all recognize the Dr human blood group antigen on the decay-accelerating factor (DAF or CD55). These adhesins are encoded by a family of closely related operons, the first characterized and sequenced being the afa operon. Consequently, it has been suggested that this group of DAEC strains producing such adhesins be named the Afa/Dr DAEC family. Three distinct but closely related gene clusters coding for phenotypically and morphologically distinct aggregative adherence fimbriae (AAF) have been characterized. In each case, electron microscopy revealed that bacterial surfaces were surrounded by long, relatively flexible fimbrial structures.

Enterohemorrhagic Escherichia coli (EHEC) was first recognized as a cause of human disease in 1983 and is associated with diarrhea and hemorrhagic colitis, which may be complicated by life-threatening renal and neurological sequelae. EHEC are defined by their ability to produce one or more Shiga-like toxins (Stx), which mediate the systemic complications of EHEC infections, and to induce characteristic attaching and effacing lesions on intestinal epithelia, a phenotype that depends on the locus of enterocyte effacement. Acquisition of Stx-encoding bacteriophages by enteropathogenic E. coli is believed to have contributed to the evolution of EHEC, and consequently some virulence factors are conserved in both pathotypes. A key requirement for E. coli to colonize the intestines and produce disease is the ability to adhere to epithelial cells lining the gastrointestinal tract. Here, we review knowledge of the adhesins produced by EHEC and other Stx-producing E. coli, with emphasis on genetic, structural, and mechanistic aspects and their contribution to pathogenesis.

Enteropathogenic Escherichia coli (EPEC) strains induce morphological changes in infected epithelial cells. The resulting attaching and effacing (A/E) lesion is characterized by intimate bacterial adherence to epithelial cells, with microvillus destruction, cytoskeletal rearrangement, and aggregation of host cytoskeletal proteins. This review presents an overview of the adhesion mechanisms used for the colonization of the human gastrointestinal tract by EPEC. The mechanisms underlying EPEC adhesion, prior to and during the formation of the A/E lesion, and the host cytosolic responses to bacterial infection leading to diarrheal disease are discussed.

This review is primarily concerned with the first step in biofilm formation, namely, bacterial attachment to surfaces. It describes three examples of bacterial adhesins, each of which belongs to a different subgroup and follows different strategies for surface presentation and adhesin exposure. These are type 1 fimbriae, very long stiff rodlike organelles; curli, amorphous fluffy coat structures; and finally antigen 43, short outer membrane structures with a simple assembly system. Their role as adhesins, their structure and biosynthesis, and their role in biofilm formation are described in detail in the review. The FimH protein presented by type 1 fimbriae seems to be a highly versatile adhesin fulfilling a diverse spectrum of roles ranging from pellicle and biofilm formation to being a bona fide virulence factor in uropathogenic E. coli (UPEC) strains, where it plays important roles in the manifestation of cystitis. Curli formation promotes two fundamental processes associated with biofilm formation: initial adhesion and cell-to-cell aggregation. A role for curli in the colonization of inert surfaces has been demonstrated. Severe sepsis and septic shock are frequently caused by gram-negative bacteria, and several factors suggest a significant role for curli during E. coli sepsis. The protection provided by Ag43-mediated aggregation was underlined in a series of experiments addressing the role of Ag43 in protection against oxidizing agents. Type 1 fimbriae, curli, and Ag43 are structurally different bacterial surface structures and follow completely different strategies for surface display and assembly.

Gram-negative bacteria assemble a variety of surface structures, including the hair-like organelles known as pili or fimbriae. Pili typically function in adhesion and mediate interactions with various surfaces, with other bacteria, and with other types of cells such as host cells. The chaperone/usher (CU) pathway assembles a widespread class of adhesive and virulence-associated pili. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and integral outer membrane protein termed the usher, which forms a multifunctional assembly and secretion platform. This review addresses the molecular and biochemical aspects of the CU pathway in detail, focusing on the type 1 and P pili expressed by uropathogenic Escherichia coli as model systems. We provide an overview of representative CU pili expressed by E. coli and Salmonella, and conclude with a discussion of potential approaches to develop antivirulence therapeutics that interfere with pilus assembly or function.

Toxins and Secretion Systems

The Escherichia coli hemolysin, earlier referred to as the hemolysin, is the best-characterized repeats in toxin (RTX) secreted by a type I exoprotein secretion system. The E. coli hemolysin is a significant virulence factor in murine models of peritonitis and ascending urinary tract infection, which suggests it is likely to be an important cytotoxin in human, extraintestinal E. coli diseases. Among E. coli or Salmonella strains there are no known examples of strict RTX leukotoxins in which lytic activity is limited to white blood cells. The general gene organization of the Vibrio cholerae RTX locus is similar to that seen with either of the E. coli hly and ehx loci with C, B, and D RTX homologs, clearly indicating it is a member of the RTX family. The hemolysin occurs less frequently in cystitis strains and only rarely among normal fecal strains. Among the extraintestinal E. coli isolates, the hlyCABDgenes were among the first virulence factors localized to unique, tRNA-associated segments of E. coli chromosomes. The hemolysin genes were eventually linked to P-type pilin and cytotoxic necrotizing factor-1 genes. Recent progress with its study has slowed down because of the difficulty in deriving the physical structure of the hemolysin protein or other RTX toxins and establishing its precise cytotoxic mechanism and role in pathogenesis of extraintestinal E. coli disease. Genomic sequencing has revealed that there are additional RTX-like genes found among many different pathogens; perhaps new efforts to discover their functions will aid progress in the RTX toxin field.

This review focuses on the function of the Escherichia coli and Salmonella autotransporters for which a considerable amount of literature is available. Members of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) family are proteins from E. coli and Shigella spp., which, like the Neisseria and Haemophilus influenzae IgA1 proteases and Hap, possess a consensus serine protease motif. The largest subfamily of autotransporters is defined by the AidA conserved domain COG3468 and consists of members from a diverse range of animal and plant pathogens including E. coli, S. enterica, Yersinia pestis. This subfamily, which is composed of more than 55 proteins, possesses some of the best-characterized autotransporter proteins including the S. flexneri mediator of motility IcsA, the major phase-variable E. coli outer membrane protein antigen 43 (Ag43) and the diffuse adhering E. coli (DAEC) adhesin AIDA-I, from which this subfamily derives its name. Another member of the AIDA-I family, and one of the most studied autotransporter proteins, is IcsA. The autotransporter pathway is emerging as the most common mechanism of protein translocation across the gram-negative outer membrane.

This article reviews the Escherichia coli toxins called cytotoxic necrotizing factors (CNFs), which cause activation of Rho GTPases. It describes their modes of action, structure-function relationships, and roles in disease. Rho GTPases, the targets of CNFs, belong to the Ras superfamily of low molecular mass GTPases and act as molecular switches in various signaling pathways. Low molecular mass GTPases of the Rho family are known as master regulators of the actin cytoskeleton. Moreover, they are involved in various signal transduction processes, from transcriptional activation, cell cycle progression, and cell transformation to apoptosis. CNFs are cytotoxic for a wide variety of cells, including 3T3 fibroblasts, Chinese hamster ovary cells, Vero cells, HeLa cells, and cell lines of neuronal origin. This implies that a commonly expressed receptor is responsible for the uptake of CNF1. Cultured mammalian cells treated with CNFs are characterized by dramatic changes in actin-containing structures, including stress fibers, lamellipodia, and filopodia. Most striking is the formation of multinucleation in these cells. Rho GTPases are increasingly recognized as essential factors in the development of cancer and metastasis. This fact has initiated a discussion as to whether activation of Rho proteins by CNFs might be involved in tumorigenesis. Moreover, CNF1 increases the expression of the cyclooxygenase 2 (Cox2) gene in fibroblasts. Increased expression of Cox2 is observed in some types of tumors, e.g., colon carcinoma. Lipid-mediators produced by the enzyme are suggested to be responsible for tumor progression.

Heat-labile enterotoxins (LTs) of Escherichia coli are closely related to cholera toxin (CT), which was originally discovered in 1959 in culture filtrates of the gram-negative bacterium Vibrio cholerae. Several other gram-negative bacteria also produce enterotoxins related to CT and LTs, and together these toxins form the V. cholerae-E. coli family of LTs. Strains of E. coli causing a cholera-like disease were designated enterotoxigenic E. coli (ETEC) strains. The majority of LTI genes (elt) are located on large, self-transmissible or mobilizable plasmids, although there are instances of LTI genes being located on chromosomes or carried by a lysogenic phage. The stoichiometry of A and B subunits in holotoxin requires the production of five B monomers for every A subunit. One proposed mechanism is a more efficient ribosome binding site for the B gene than for the A gene, increasing the rate of initiation of translation of the B gene independently from A gene translation. The three-dimensional crystal structures of representative members of the LT family (CT, LTpI, and LTIIb) have all been determined by X-ray crystallography and found to be highly similar. Site-directed mutagenesis has identified many residues in the CT and LT A subunits, including His44, Val53, Ser63, Val97, Glu110, and Glu112, that are critical for the structures and enzymatic activities of these enterotoxins. For the enzymatically active A1 fragment to reach its substrate, receptor-bound holotoxin must gain access to the cytosol of target cells.

The Shiga toxins (Stxs), also known as Vero toxins and previously called Shiga-like toxins, are a family of potent protein synthesis inhibitors made by Shigella dysenteriae type 1 and some serogroups of Escherichia coli that cause bloody diarrhea in humans. Stxs act as virulence factors for both S. dysenteriae and E. coli and contribute to the disease process initiated by those organisms both directly and indirectly. A handful of methods exist for toxin purification, and the toxins can now even be purchased commercially. However, the Stxs are now classified as select agents, and specific rules govern the distribution of both the toxin and clones of the toxin. Toxin delivery into the host in S. dysenteriae type 1 is most likely aided by the invasiveness of that organism. Although the Stxs are made and produced by bacteria, they do not appear to act against either their host organism or other bacteria under normal circumstances, most likely because the A subunit is secreted from the cytoplasm as soon as it is synthesized and because the holotoxin cannot enter intact bacterial cells. The effectiveness of antibiotic therapy in patients infected with Stx-producing E. coli (STEC) such as O157:H7 as well as the potential risks of such treatment are areas of controversy. Several studies indicate that the course of the diarrhea stage of the disease is unaltered by antibiotic treatment. Several groups anticipate that a therapy that targets the Stxs is an important component of trying to alleviate disease caused by Stx-producing bacteria.

While the DNA damage induced by ionizing radiation and by many chemical compounds and drugs is well characterized, the genotoxic insults inflicted by bacteria are only scarcely documented. However, accumulating evidence indicates that we are exposed to bacterial genotoxins. The prototypes of such bacterial genotoxins are the Cytolethal Distending Toxins (CDTs) produced by Escherichia coli and Salmonella enterica serovar Typhi. CDTs display the DNase structure fold and activity, and induce DNA strand breaks in the intoxicated host cell nuclei. E. coli and certain other Enterobacteriaceae species synthesize another genotoxin, colibactin. Colibactin is a secondary metabolite, a hybrid polyketide/nonribosomal peptide compound synthesized by a complex biosynthetic machinery. In this review, we summarize the current knowledge on CDT and colibactin produced by E. coli and/or Salmonella Typhi. We describe their prevalence, genetic determinants, modes of action, and impact in infectious diseases or gut colonization, and discuss the possible involvement of these genotoxigenic bacteria in cancer.

Escherichia colicauses three types of illnesses in humans: diarrhea, urinary tract infections, and meningitis in newborns. The acquisition of virulence-associated genes and the ability to properly regulate these, often horizontally transferred, loci distinguishes pathogens from the normally harmless commensal E. coli found within the human intestine. This review addresses our current understanding of virulence gene regulation in several important diarrhea-causing pathotypes, including enteropathogenic, enterohemorrhagic,enterotoxigenic, and enteroaggregativeE. coli—EPEC, EHEC, ETEC and EAEC, respectively. The intensely studied regulatory circuitry controlling virulence of uropathogenicE. coli, or UPEC, is also reviewed, as is that of MNEC, a common cause of meningitis in neonates. Specific topics covered include the regulation of initial attachment events necessary for infection, environmental cues affecting virulence gene expression, control of attaching and effacing lesionformation, and control of effector molecule expression and secretion via the type III secretion systems by EPEC and EHEC. How phage control virulence and the expression of the Stx toxins of EHEC, phase variation, quorum sensing, and posttranscriptional regulation of virulence determinants are also addressed. A number of important virulence regulators are described, including the AraC-like molecules PerA of EPEC, CfaR and Rns of ETEC, and AggR of EAEC;the Ler protein of EPEC and EHEC;RfaH of UPEC;and the H-NS molecule that acts to silence gene expression. The regulatory circuitry controlling virulence of these greatly varied E. colipathotypes is complex, but common themes offerinsight into the signals and regulators necessary forE. coli disease progression.

Shigella species are the causative agents of bacillary dysentery in humans, an invasive disease in which the bacteria enter the cells of the epithelial layer of the large intestine, causing extensive tissue damage and inflammation. They rely on a plasmid-encoded type III secretion system (TTSS) to cause disease; this system and its regulation have been investigated intensively at the molecular level for decades. The lessons learned have not only deepened our knowledge of Shigella biology but also informed in important ways our understanding of the mechanisms used by other pathogenic bacteria to cause disease and to control virulence gene expression. In addition, the Shigella story has played a central role in the development of our appreciation of the contribution of horizontal DNA transfer to pathogen evolution.A 30-kilobase-pair "Entry Region" of the 230-kb virulence plasmid lies at the heart of the Shigella pathogenesis system. Here are located the virB and mxiE regulatory genes and most of the structural genes involved in the expression of the TTSS and its effector proteins. Expression of the virulence genes occurs in response to an array of environmental signals, including temperature, osmolarity, and pH.At the top of the regulatory hierarchy and lying on the plasmid outside the Entry Region isvirF, encoding an AraC-like transcription factor.Virulence gene expression is also controlled by chromosomal genes,such as those encoding the nucleoid-associated proteins H-NS, IHF, and Fis, the two-component regulators OmpR/EnvZ and CpxR/CpxA, the anaerobic regulator Fnr, the iron-responsive regulator Fur, and the topoisomerases of the cell that modulate DNA supercoiling. Small regulatory RNAs,the RNA chaperone Hfq,and translational modulation also affect the expression of the virulence phenotypetranscriptionally and/orposttranscriptionally.

σN (also σ54) is an alternative sigma factor subunit of the RNA polymerase complex that regulates the expression of genes from many different ontological groups. It is broadly conserved in the Eubacteria with major roles in nitrogen metabolism, membrane biogenesis, and motility. σN is encoded as the first gene of a five-gene operon including rpoN (σN), ptsN, hpf, rapZ, and npr that has been genetically retained among species of Escherichia, Shigella, and Salmonella. In an increasing number of bacteria, σN has been implicated in the control of genes essential to pathogenic behavior, including those involved in adherence, secretion, immune subversion, biofilm formation, toxin production, and resistance to both antimicrobials and biological stressors. For most pathogens how this is achieved is unknown. In enterohemorrhagic Escherichia coli (EHEC) O157, Salmonella enterica, and Borrelia burgdorferi, regulation of virulence by σN requires another alternative sigma factor, σS, yet the model by which σN-σS virulence regulation is predicted to occur is varied in each of these pathogens. In this review, the importance of σN to bacterial pathogenesis is introduced, and common features of σN-dependent virulence regulation discussed. Emphasis is placed on the molecular mechanisms underlying σN virulence regulation in E. coli O157. This includes a review of the structure and function of regulatory pathways connecting σN to virulence expression, predicted input signals for pathway stimulation, and the role for cognate σN activators in initiation of gene systems determining pathogenic behavior.

Salmonellosis, caused by members of the genus Salmonella, is responsible for considerable global morbidity and mortality in both animals and humans. In this review, we will discuss the pathogenesis of Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium, focusing on human Salmonella infections. We will trace the path of Salmonella through the body, including host entry sites, tissues and organs affected, and mechanisms involved in both pathogenesis and stimulation of host immunity. Careful consideration of the natural progression of disease provides an important context in which attenuated live oral vaccines can be rationally designed and developed. With this in mind, we will describe a series of attenuated live oral vaccines that have been successfully tested in clinical trials and demonstrated to be both safe and highly immunogenic. The attenuation strategies summarized in this review offer important insights into further development of attenuated vaccines against other Salmonella for which live oral candidates are currently unavailable.

Eukaryotes have evolved strategies to detect microbial intrusion and instruct immune responses to limit damage from infection. Recognition of microbes and cellular damage relies on the detection of microbe-associated molecular patterns (MAMPs, also called PAMPS, or pathogen-associated molecular patterns) and so-called "danger signals" by various families of host pattern recognition receptors (PRRs). Members of the recently identified protein family of nucleotide-binding domain andleucine-rich-repeat-containing proteins (NLR), including Nod1, Nod2, NLRP3, and NLRC4, have been shown to detect specific microbial motifs and danger signals for regulating host inflammatory responses. Moreover, with the discovery that polymorphisms in NOD1, NOD2, NLRP1, and NLRP3 are associated with susceptibility to chronic inflammatory disorders, the view has emerged that NLRs act not only as sensors butalso can serve as signaling platforms for instructing and balancing host immune responses. In this chapter, we explore the functions of these intracellular innate immune receptors and examine their implication in inflammatory diseases.

The host-pathogen interaction involves a myriad of initiations and responses from both sides. Bacterial pathogens such as enteropathogenic Escherichia coli (EPEC) and Salmonella enterica have numerous virulence factors that interact with and alter signaling components of the host cell to initiate responses that are beneficial to pathogen survival and persistence. The study of Salmonella and EPEC infection reveals intricate connections between host signal transduction, cytoskeletal architecture, membrane trafficking, and cytokine gene expression. The emerging picture includes elements of molecular mimicry by bacterial effectors and bacterial subversion of typical host events, with the result that EPEC is able to survive and persist in an extracellular milieu, while Salmonella establishes an intracellular niche and is able to spread systemically throughout the host. This review focuses on recent advances in our understanding of the signaling events stemming from the host-pathogen interactions specific to Salmonella and EPEC.

The recruitment and activation of phagocytic cells in infected tissues and the induction of T-cell- and B-cell-dependent acquired immunity are crucial for the control and resolution of Salmonella infections. These complex processes require the interaction of bacteria with a multitude of cell surface receptors and the controlled production of soluble mediators. The mechanisms of cytokine induction in response to Salmonella and the role of cytokine networks in Salmonella infections are the main foci of this review. Pathogen-associated molecular pattern receptors play an important role in recognition of bacteria by the host. Effective immunity against the bacterium therefore relies on the ability of the host to recruit phagocytes in the tissues and to enhance the antibacterial functions of these inflammatory cells. TNF-a, IFN-?, IL12, IL15, and IL18 are needed for the full expression of innate host resistance to Salmonella. The genes for mammalian cytokines can be cloned into suitable vectors and expressed in Salmonella as functional proteins. The in vivo production of cytokines by Salmonella carriers can have therapeutic applications and can modulate immune functions in the host. The possibility to modulate antigen-specific immune responses by expressing cytokines in Salmonella is illustrated by the increase in Salmonella-specific IgA responses induced by administration of IL-5-expressing bacteria. The same cytokines that are responsible for endotoxic shock are elevated in the late stages of lethal Salmonella infections, indicating that the toxicity of Salmonella lipopolysaccharide (LPS) may actually be contributing to the death of the host.

This review discusses the role that nitric oxide (NO) and its congeners play on various stages in the pathophysiology of Escherichia coli and Salmonella infections, with special emphasis on the regulatory pathways that lead to high NO synthesis, the role of reactive nitrogen species (RNS) in host resistance, and the bacterial molecular targets and defense mechanisms that protect enteric bacteria against the nitrosative stress encountered in diverse host anatomical sites. In general, NO can react directly with prosthetic groups containing transition metal centers, with other radicals, or with sulfhydryl groups in the presence of an electron acceptor. Binding to iron complexes is probably the best characterized direct reaction of NO in biological systems. The targets of RNS are numerous. RNS can facilitate oxidative modifications including lipid peroxidation, hydroxylation, and DNA base and protein oxidation. In addition, RNS can inflict nitrosative stress through the nitrosation of amines and sulfhydryls. Numerous vital bacterial molecules can be targeted by NO. It is therefore not surprising that enteropathogenic bacteria are armed with a number of sensors to coordinate the protective response to nitrosative stress, along with an assortment of antinitrosative defenses that detoxify, repair, or avoid the deleterious effects of RNS encountered within the host. NO and NO-derived RNS play important roles in innate immunity to Salmonella and E. coli. Enzymatic NO production by NO synthases can be enhanced by microbial and other inflammatory stimuli and it exerts direct antimicrobial actions as well as immunomodulatory and vasoregulatory effects.

This review summarizes recent data on iron metabolism in macrophages, with a special emphasis on possible bacteriostatic and bactericidal consequences for intracellular pathogens. It includes the role of biological chelators and transporters in normal macrophage physiology and antimicrobial defense. Iron is an essential metal cofactor for many biochemical pathways in mammals. However, excess iron promotes the formation of cytotoxic oxygen derivatives so that systemic iron levels must be tightly regulated. The mechanism of iron recycling by macrophages including iron efflux from erythrocyte-containing phagosomes, iron release from macrophages, and entry into the transferrin (Tf) cycle remain poorly understood. Ferroportin expression in the liver, spleen, and bone marrow cells appears to be restricted to macrophages. Mutant mice bearing a conditional deletion of the ferroportin gene in macrophages show retention of iron by hepatic Kupffer cells and splenic macrophages. Hepcidin is induced by lipopolysaccharide (LPS) in mouse spleens and splenic macrophage in vitro and appears to mediate the LPS-induced down-regulation of ferroportin in the intestine and in splenic macrophages, suggesting that inflammatory agents may regulate iron metabolism through modulation of ferroportin expression. The host transporter Nramp1 may compete directly with bacterial divalent-metal transport systems for the acquisition of divalent metals within the phagosomal space. The ultimate outcome of these competing interactions influences the ability of pathogens to survive and replicate intracellularly. This seems particularly relevant to the Salmonella, Leishmania, and Mycobacterium spp., in which inactivating mutations in Nramp1 abrogate the natural resistance of macrophages to these pathogens.

The interaction betweenSalmonella and its host is complex and dynamic: the host mounts an immune defense against the pathogen, which in turn acts to reduce, evade, or exploit these responses to successfully colonize the host. Although the exact mechanisms mediating protective immunity are poorly understood, it is known that T cells are a critical component of immunity to Salmonella infection, and a robust T-cell response is required for both clearance of primary infection and resistance to subsequent challenge. B-cell functions, including but not limited to antibody production, are also required for generation of protective immunity. Additionally, interactions among host cells are essential. For example, antigen-presenting cells (including B cells) express cytokines that participate in CD4+ T cell activation and differentiation. Differentiated CD4+ T cells secrete cytokines that have both autocrine and paracrine functions, including recruitment and activation of phagocytes, and stimulation of B cell isotype class switching and affinity maturation. Multiple bacterium-directed mechanisms, including altered antigen expression and bioavailability and interference with antigen-presenting cell activation and function, combine to modify Salmonella's "pathogenic signature" in order to minimize its susceptibility to host immune surveillance. Therefore, a more complete understanding of adaptive immune responses may provide insights into pathogenic bacterial functions. Continued identification of adaptive immune targets will guide rational vaccine development, provide insights into host functions required to resist Salmonella infection, and correspondingly provide valuable reagents for defining the critical pathogenic capabilities of Salmonella that contribute to their success in causing acute and chronic infections.

The best-characterized mucosa-associated lymphoid tissue (MALT), and also the most relevant for this review, is the gastrointestinal-associated lymphoid tissue (GALT). The review reviews our understanding of the importance of mucosal immune responses in resisting infections caused by E. coli and Salmonella spp. It focuses on the major human E. coli infections and discusses whether antigen-specific mucosal immune responses are important for resistance against primary infection or reinfection by pathogenic E. coli. It analyzes human data on mucosal immunity against E. coli, a growing body of data of mucosal responses in food production animals and other natural hosts of E. coli, and more recent experimental studies in mice carrying defined deletions in genes encoding specific immunological effectors, to show that there may be considerable conservation of the effective host mucosal immune response against this pathogen. The species Salmonella enterica contains a number of serovars that include pathogens of both humans and animals; these bacteria are frequently host specific and may cause different diseases in different hosts. Ingestion of various Salmonella serovars, such as Typhimurium, results in localized infections of the small intestine leading to gastroenteritis in humans, whereas ingestion of serovar Typhi results in systemic infection and enteric fever. Serovar Typhi infects only humans, and the review discusses the mucosal immune responses against serovar Typhi, focusing on the responses in humans and in the mouse typhoid fever model.

A clear etiological link has been established between infection with several gram-negative enteric pathogens, including Salmonella spp., and the incidence of reactive arthritis (ReA), an autoimmune disease that largely affects the joints. ReA is sometimes referred to as Reiter's syndrome, particularly when accompanied by uveitis and urethritis. This review reviews the evidence etiologically linking Salmonella infection with autoimmune disease and addresses the roles that bacterial and host elements play in controlling disease outcome. ReA is an autoimmune disease that largely consists of painful joint inflammation but also can include inflammation of the eye, gastrointestinal tract, and skin. ReA is a member of a broad spectrum of chronic inflammatory disorders termed the seronegative spondyloarthropathies (SNSpAs) that includes ankylosing spondylitis (AS), psoriatic arthritis, and enteropathic arthritis. Salmonella species, as well as other enteric pathogens associated with postgastroenteritis ReA, are facultative intracellular gram-negative bacteria. Many studies have analyzed the association of the HLA class I molecule, HLA-B27, with SNSpAs. Whereas B27 has been shown to be present in 90 to 95% of cases of AS, the association of the B27 haplotype with other SNSpAs is more tenuous. The clear association between ReA and infection with Salmonella or other gram-negative enteric pathogens has led to the suggestion that the adaptive immune response to infection has an autoimmune component. In addition to various Salmonella species, other gram-negative enteric pathogens have been linked to the development of ReA. Given their close relationship to Salmonella, this review considers the involvement of Shigella species in ReA.

Infectious diseases represent one of the most common causes of death worldwide, with the enteropathogenic bacteria Salmonella and Shigella and pathogenic Escherichia coli being among the most detrimental. Currently, vaccination represents the preferred method of preventing such infections. For stimulating the adaptive immune response, immunizations are frequently based on formulations which include inactivated whole-cell vaccines, live attenuated vaccines, or subunit vaccines. These can be administered via a parenteral or mucosal route, the latter having the advantage that it most closely mimics the actual course of infection. In addition to the type of vaccine and method of application, important consideration needs to be paid to safety, efficacy, and cost, which are often major bottlenecks in the successful implementation of vaccines. In this chapter we take a limited look at the history surrounding vaccinations involving Salmonella, Shigella, and pathogenic E. coli. Salmonella infections, which can lead to typhoid fever, are becoming increasing difficult to treat with antibiotics due to multi-drug-resistant strains. At present, the parenteral Vi-based subunit vaccines and the live attenuated oral vaccine Ty21a have proven to be the vaccines of choice, with high levels of protective efficacy and limited side effects. Shigella infections are responsible for the diarrheal disease shigellosis. Various live and nonliving mucosal and parenteral vaccines have been tested, with the most promising candidates evolving around those that stimulate the production of O-antigen-specific antibodies. Pathogenic Escherichia coli infections can lead to severe diseases due to the bacterium's production of several specific toxins. Vaccines against this bacterium target its toxins, as well as surface-exposed antigens, all of which have been found to be effective as immunogens.

This review reviews the critical role played by cytokines in the pathogenesis of Escherichia coli sepsis. It focuses on prototypic pro-inflammatory and anti-inflammatory cytokines and their influence on mortality in experimental animal models of E. coli endotoxemia and of live E. coli sepsis. The review reviews the results of clinical trials on anticytokine therapy in patients with severe sepsis or septic shock. The recognition of the critical role played by tumor necrosis factor (TNF), a secreted 17kDa cytokine, in endotoxic and gram-negative shock has been a major step forward in our understanding of the pathogenesis of sepsis. The review describes the role of TNF, IL1, and IL6 in animal models of E. coli endotoxemia and sepsis. Given the pivotal role played by TNF in experimental sepsis and the fact that elevated concentrations of TNF were detected in the circulation of patients with sepsis, anti-TNF treatment strategies were investigated as adjunctive therapy for severe sepsis and septic shock. Several studies demonstrated that high levels of interleukin-6 (IL-6) are associated with an increased risk for fatal outcome. Gamma interferon (IFN-γ), IL-12, and IL-18 are functionally related cytokines. A recent study has indicated that transgenic mice overexpressing IL-15 are resistant to an otherwise lethal intraperitoneal E. coli challenge. IL4, IL10, and IL13are prototypic anti-inflammatory cytokines. Their classification as anti-inflammatory cytokines is based on the observation that these molecules inhibit the production of proinflammatory cytokines (primarily TNF and IL1) and toxic oxygen and reactive nitrogen species by myeloid cells.

Salmonella spp. can infect host cells by gaining entry through phagocytosis or by inducing host cell membrane ruffling that facilitates bacterial uptake. With its wide host range, Salmonella enterica serovar Typhimurium has proven to be an important model organism for studying intracellular bacterial pathogenesis. Upon entry into host cells, serovar Typhimurium typically resides within a membrane-bound compartment termed the Salmonella-containing vacuole (SCV). From the SCV, serovar Typhimurium can inject several effector proteins that subvert many normal host cell systems, including endocytic trafficking, cytoskeletal rearrangements, lipid signaling and distribution, and innate and adaptive host defenses. The study of these intracellular events has been made possible through the use of various imaging techniques, ranging from classic methods of transmission electron microscopy to advanced livecell fluorescence confocal microscopy. In addition, DNA microarrays have now been used to provide a "snapshot" of global gene expression in serovar Typhimurium residing within the infected host cell. This review describes key aspects of Salmonella-induced subversion of host cell activities, providing examples of imaging that have been used to elucidate these events. Serovar Typhimurium engages specific host cell machinery from initial contact with the host cell to replication within the SCV. This continuous interaction with the host cell has likely contributed to the extensive arsenal that serovar Typhimurium now possesses, including two type III secretion systems, a range of ammunition in the form of TTSS effectors, and a complex genetic regulatory network that coordinates the expression of hundreds of virulence factors.

Infectious diseases are among the leading causes of mortality worldwide, and numerous bacterial species are included in the vast array of causative agents. This review describes microscopy-based techniques that can be used to study interactions between bacteria and infected host cells, bacterial gene expression in the infected animal, and bacteria-induced cell signaling in eukaryotic cells. As infectious model systems, urinary tract infections caused by uropathogenic Escherichia coli (UPEC) and a mouse model of typhoid fever caused by Salmonella enterica serovar Typhimurium are used. To study the interaction mechanism between bacteria and eukaryotic cells, one commonly uses cell lines, primary cells, and animal models. Within the host, bacteria can be located in various organs where they are exposed to different cell types, ranging from epithelial cells at the mucosal linings to phagocytic white blood cells. In each site, bacteria are exposed to specific sets of innate immune defense mechanisms, and to survive these threats, bacteria must rapidly adapt their gene expression profile to maximize their chance of survival in any situation. The rapid development of fluorescent reporter proteins and advances in microscopy-based techniques have provided new and promising approaches not only to locate bacteria in tissues, but also to analyze expression of virulence factors in individual bacteria and host cells during the progression of disease. These techniques enable, for the first time, studies of the complex microenvironments within infected organs and will reveal the alterations of bacterial physiology that occur during bacterial growth within a host.

Extraintestinal Infections

Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis contributes to such mortality and morbidity. Recent reports of E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. Studies using in vitro and in vivo models of the blood-brain barrier have shown that E. coli meningitis follows a high degree of bacteremia and invasion of the blood-brain barrier. E. coli invasion of the blood-brain barrier, the essential step in the development of E. coli meningitis, requires specific microbial and host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors contributing to E. coli invasion of the blood-brain barrier is shown to be efficient in preventing E. coli penetration into the brain. The basis for requiring a high degree of bacteremia for E. coli penetration of the blood-brain barrier, however, remains unclear. Continued investigation on the microbial and host factors contributing to a high degree of bacteremia and E. coli invasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis.

The urinary tract is among the most common sites of bacterial infection, and Escherichia coli is by far the most common species infecting this site. Individuals at high risk for symptomatic urinary tract infection (UTI) include neonates, preschool girls, sexually active women, and elderly women and men. E. coli that cause the majority of UTIs are thought to represent only a subset of the strains that colonize the colon. E. coli strains that cause UTIs are termed uropathogenic E. coli (UPEC). In general, UPEC strains differ from commensal E. coli strains in that the former possess extragenetic material, often on pathogenicity-associated islands (PAIs), which code for gene products that may contribute to bacterial pathogenesis. Some of these genes allow UPEC to express determinants that are proposed to play roles in disease. These factors include hemolysins, secreted proteins, specific lipopolysaccharide and capsule types, iron acquisition systems, and fimbrial adhesions. The current dogma of bacterial pathogenesis identifies adherence, colonization, avoidance of host defenses, and damage to host tissues as events vital for achieving bacterial virulence. These considerations, along with analysis of the E. coli CFT073, UTI89, and 536 genomes and efforts to identify novel virulence genes should advance the field significantly and allow for the development of a comprehensive model of pathogenesis for uropathogenic E. coli.Further study of the adaptive immune response to UTI will be especially critical to refine our understanding and treatment of recurrent infections and to develop vaccines.

Extraintestinal pathogenic Escherichia coli (ExPEC) are important pathogens in humans and certain animals. Molecular epidemiological analyses of ExPEC are based on structured observations of E. coli strains as they occur in the wild. By assessing real-world phenomena as they occur in authentic contexts and hosts, they provide an important complement to experimental assessment. Fundamental to the success of molecular epidemiological studies are the careful selection of subjects and the use of appropriate typing methods and statistical analysis. To date, molecular epidemiological studies have yielded numerous important insights into putative virulence factors, host-pathogen relationships, phylogenetic background, reservoirs, antimicrobial-resistant strains, clinical diagnostics, and transmission pathways of ExPEC, and have delineated areas in which further study is needed. The rapid pace of discovery of new putative virulence factors and the increasing awareness of the importance of virulence factor regulation, expression, and molecular variation should stimulate many future molecular epidemiological investigations. The growing sophistication and availability of molecular typing methodologies, and of the new computational and statistical approaches that are being developed to address the huge amounts of data that whole genome sequencing generates, provide improved tools for such studies and allow new questions to be addressed.

This review reviews the pathogenesis of different phases of Salmonella infections. The nature of Salmonella infections in several domesticated animal species is described to highlight differences in the epidemiology and pathogenesis of salmonellosis in different hosts. The biology of Salmonella serovar host specificity is discussed in the context of our current understanding of the molecular basis of pathogenesis and the potential impact of different virulence determinants on Salmonella natural history. The ability to colonize the intestine, as evidenced by the shedding of relatively large numbers of bacteria in the feces over a long period, is shared unequally by Salmonella serovars. Studies probing the molecular basis of Salmonella intestinal colonization have been carried out by screening random transposon mutant banks of serovar Typhimurium in a range of avian and mammalian species. It is becoming increasingly clear that Salmonella pathogenicity island 2 (SPI2) is a major virulence factor during infection of food-producing animals, including cattle and poultry. The prevalence of Salmonella serovars in domestic fowl varies in different countries and with time. Although chickens are the natural hosts of serovars Gallinarum and Pullorum, natural outbreaks caused by these serovars in turkeys, guinea fowl, and other avian species have been described. There are two possible explanations to account for the apparent host specificity of certain Salmonella serovars. Environmental factors may increase exposure of particular animal species to certain serovars. Alternatively, there are genetic differences between these serovars, which allow them to survive and/or grow in specific niches only found within ruminants or pigs.

Human salmonellosis is generally associated with Salmonella enterica from subspecies enterica (subspecies I). Acute infections can present in one of four ways: enteric fever, gastroenteritis, bacteremia, or extraintestinal focal infection. As with other infectious diseases, the course and outcome of the infection depend on a variety of factors, including the infecting organism, the inoculating dose, and the immune status and genetic background of the host. For serovarsTyphi and Paratyphi A there is a clear association between the genetic background of the serovar and systemic infection in humans. For serovars Paratyphi B and Paratyphi C, a good clinical description of the host and detailed population genetics of the pathogen are necessary before more detailed genetic studies of novel virulence factors,or host factors,can be initiated. For the nontyphoidalserovars (NTS) the situation is less clear. Serovars Typhimurium and Enteritidis are the most common within the food chain, and so the large number of invasive infections associated with these serovars is most likely due to exposure rather than to increased virulence of the pathogen. In Africa, however, a closely related group of strains of serovar Typhimurium, associated with HIV infection, may have become host adapted tohumans, suggesting that not all isolates called "Typhimurium" should be considered as a single group. Here we review current knowledge of the salmonellae for which invasive disease in humans is an important aspect of their population biology.

Proteus mirabilis, a Gram-negative rod-shaped bacterium most noted for its swarming motility and urease activity, frequently causes catheter-associated urinary tract infections (CAUTIs) that are often polymicrobial. These infections may be accompanied by urolithiasis, the development of bladder or kidney stones due to alkalinization of urine from urease-catalyzed urea hydrolysis. Adherence of the bacterium to epithelial and catheter surfaces is mediated by 17 different fimbriae, most notably MR/P fimbriae. Repressors of motility are often encoded by these fimbrial operons. Motility is mediated by flagella encoded on a single contiguous 54-kb chromosomal sequence. On agar plates, P. mirabilis undergoes a morphological conversion to a filamentous swarmer cell expressing hundreds of flagella. When swarms from different strains meet, a line of demarcation, a “Dienes line,” develops due to the killing action of each strain’s type VI secretion system. During infection, histological damage is caused by cytotoxins including hemolysin and a variety of proteases, some autotransported. The pathogenesis of infection, including assessment of individual genes or global screens for virulence or fitness factors has been assessed in murine models of ascending urinary tract infections or CAUTIs using both single-species and polymicrobial models. Global gene expression studies performed in culture and in the murine model have revealed the unique metabolism of this bacterium. Vaccines, using MR/P fimbria and its adhesin, MrpH, have been shown to be efficacious in the murine model. A comprehensive review of factors associated with urinary tract infection is presented, encompassing both historical perspectives and current advances.

In this chapter we review the literature with respect to what is known about how Escherichia coli colonizesthe mammalian intestine. We begin with a brief discussion of the mammalian large intestine, the major site that commensal strains of E. coli colonize. Next, evidence is discussed showing that, in order to colonize, E. coli must be able to penetrate and grow in the mucus layer of the large intestine. This is followed by discussions of colonization resistance, i.e., factors that are involved in the ability of a complete microbiota (microflora) to resist colonization by an invading bacterium, the advantages and disadvantages of the in vivo colonization models used in colonization research, the initiation and maintenance stages of E. coli colonization, and the rate of E. coli growth in the intestine. The next two sections of the chapter discuss the role of motility in colonization and how adhesion to mucosal receptors aids or inhibits penetration of the intestinal mucus layer and thereby either promotes or prevents E. coli colonization. Finally, the contribution of nutrition to the ability of E. coli to colonize is discussed based on the surprising finding that different nutrients are used by E. coli MG1655, a commensal strain, and by E. coli EDL933, an enterohemorrhagic strain, to colonize the intestine.

E. coli is a relevant model organism for the study of the molecular mechanisms underlying surface colonization. This process requires two essential steps: adhesion to a surface, followed by cell-cell adhesion counteracting the shear forces of the environment, with both steps contributing to the formation of a biofilm. This review provides an overview of the current knowledge of the genetic analyses aiming at identifying factors involved in both of these two highly related biological processes, with a particular emphasis on studies performed in Escherichia coli K-12. Bacterial adhesion to abiotic surfaces is likely to be highly dependent on the physicochemical and electrostatic interactions between the bacterial envelope and the substrate, which is itself often conditioned by the fluids to which it is exposed. Genetic analyses have revealed the diversity of genetic factors in E. coli that participate in colonization and biofilm formation on abiotic surfaces. The study of surface colonization and biofilm formation represents a rapidly expanding field of investigation. The use of E. coli K-12 to investigate the genetic basis of bacterial interactions with surfaces has led to the identification of a large repertoire of adhesins whose expression is subject to a complex interplay between regulatory networks. Understanding how E. coli K-12 behaves in complex biofilm communities will certainly contribute to an understanding of how natural commensal and pathogenic E. coli isolates develop.

Bacteria must be able to respond rapidly to changes in the environment to survive. One means of coordinating gene expression relies on tightly regulated and complex signaling systems. One of the first signaling systems that was described in detail is quorum sensing (QS). During QS, a bacterial cell produces and secretes a signaling molecule called an autoinducer (AI). As the density of the bacterial population increases, so does the concentration of secreted AI molecules, thereby allowing a bacterial species to coordinate gene expression based on population density. Subsequent studies have demonstrated that bacteria are also able to detect signal molecules produced by other species of bacteria as well as hormones produced by their mammalian hosts. This type of signaling interaction has been termed cell-to-cell signaling because it does not rely on a threshold concentration of bacterial cells. This review discusses the three main types of cell-to-cell signaling mechanisms used by Escherichia coli and Salmonella: the LuxR process, in which E. coli and Salmonella detect signals produced by other species of bacteria; the LuxS/AI-2 system, in which E. coli and Salmonella participate in intra- and interspecies signaling; and the AI-3/epinephrine/norepinephrine system, in which E. coli and Salmonella recognize self-produced AI, signal produced by other microbes, and/or the human stress hormones epinephrine and/or norepinephrine.

Biochemical network reconstructions have become popular tools in systems biology. Metabolicnetwork reconstructions are biochemically, genetically, and genomically (BiGG) structured databases of biochemical reactions and metabolites. They contain information such as exact reaction stoichiometry, reaction reversibility, and the relationships between genes, proteins, and reactions. Network reconstructions have been used extensively to study the phenotypic behavior of wild-type and mutant stains under a variety of conditions, linking genotypes with phenotypes. Such phenotypic simulations have allowed for the prediction of growth after genetic manipulations, prediction of growth phenotypes after adaptive evolution, and prediction of essential genes. Additionally, because network reconstructions are organism specific, they can be used to understand differences between organisms of species in a functional context.There are different types of reconstructions representing various types of biological networks (metabolic, regulatory, transcription/translation). This chapter serves as an introduction to metabolic and regulatory network reconstructions and models and gives a complete description of the core Escherichia coli metabolic model. This model can be analyzed in any computational format (such as MATLAB or Mathematica) based on the information given in this chapter. The core E. coli model is a small-scale model that can be used for educational purposes. It is meant to be used by senior undergraduate and first-year graduate students learning about constraint-based modeling and systems biology. This model has enough reactions and pathways to enable interesting and insightful calculations, but it is also simple enough that the results of such calculations can be understoodeasily.

EcoCyc is a bioinformatics database available at EcoCyc.org that describes the genome and the biochemical machinery of Escherichia coli K-12 MG1655. The long-term goal of the project is to describe the complete molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists and for biologists who work with related microorganisms. The database includes information pages on each E. coli gene, metabolite, reaction, operon, and metabolic pathway. The database also includes information on E. coli gene essentiality and on nutrient conditions that do or do not support the growth of E. coli. The website and downloadable software contain tools for analysis of high-throughput data sets. In addition, a steady-state metabolic flux model is generated from each new version of EcoCyc. The model can predict metabolic flux rates, nutrient uptake rates, and growth rates for different gene knockouts and nutrient conditions. This review provides a detailed description of the data content of EcoCyc and of the procedures by which this content is generated.

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

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About EcoSal Plus

EcoSal Plus is the authoritative online review journal that publishes an ever-growing body of expert reviews covering virtually all
aspects of E. coli, Salmonella, and other members of the
family Enterobacteriaceae and their
use as model microbes for biological explorations. This journal is intended
primarily for the research community as a comprehensive and
continuously updated archive of the entire corpus of knowledge about the
enteric bacterial cell. Thoughtful reviews focus on physiology, metabolism, genetics,
pathogenesis, ecology, genomics, systems biology, and history E. coli and its relatives. These provide the integrated background needed for most microbiology
investigations and are essential reading for research scientists. Articles contain links to E. coli K12 genes on the EcoCyc database site and are available as downloadable PDF files. Images and tables are downloadable to PowerPoint files. For subscription information, see the Home page. EcoSal Plus is indexed in the PubMed, CAS, Scopus, and AGRICOLA databases.

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